Nucleic acid and corresponding protein entitled 151p3d4 useful in treatment and detection of cancer

ABSTRACT

A novel gene (designated 151P3D4) and its encoded protein, and variants thereof, are described wherein 151P3D4 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 151P3D4 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 151P3D4 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 151P3D4 can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/833,918 filed Aug.3, 2007, now allowed, which is a continuation of U.S. Ser. No.10/120,907 filed Apr. 9, 2002, now abandoned, which claims priority fromU.S. Ser. No. 60/282,739 filed Apr. 10, 2001, and U.S. Ser. No.60/286,630 filed Apr. 25, 2001. The contents of these applications arehereby incorporated by reference herein in their entirety.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS WEB

This application is being filed electronically via the USPTO EFS-WEBserver, as authorized and set forth in MPEP §1730 II.B.2(a)(A), and thiselectronic filing includes an electronically submitted sequence (SEQ ID)listing. The entire content of this sequence listing is hereinincorporated by reference for all purposes. The sequence listing isidentified on the electronically filed .txt file as follows:

File Name Date of Creation Size (bytes) 511582006910Seqlist.txt Jan. 8,2010 210,345 bytes

FIELD OF THE INVENTION

The invention described herein relates to a gene and its encodedprotein, termed 151P3D4, expressed in certain cancers, and to diagnosticand therapeutic methods and compositions useful in the management ofcancers that express 151P3D4.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res1996 September 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl AcadSci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen(PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and 8 per 100,000 in women. The historic male/femaleratio of 3:1 may be decreasing related to smoking patterns in women.There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800in men and 3,900 in women). Bladder cancer incidence and mortalitystrongly increase with age and will be an increasing problem as thepopulation becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 151P3D4, that hasnow been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of 151P3D4 gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of151P3D4 are provided. The tissue-related profile of 151P3D4 in normaladult tissues, combined with the over-expression observed in the tissueslisted in Table I, shows that 151P3D4 is aberrantly over-expressed in atleast some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 151P3D4 genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding151P3D4-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a151P3D4-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the151P3D4 genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides that hybridize to the 151P3D4 genes, mRNAs, or to151P3D4-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 151P3D4. Recombinant DNA moleculescontaining 151P3D4 polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of 151P3D4gene products are also provided. The invention further providesantibodies that bind to 151P3D4 proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker ortherapeutic agent. In certain embodiments there is a proviso that theentire nucleic acid sequence of FIG. 2 is not encoded and/or the entireamino acid sequence of FIG. 2 is not prepared. In certain embodiments,the entire nucleic acid sequence of FIG. 2 is encoded and/or the entireamino acid sequence of FIG. 2 is prepared, either of which are inrespective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of 151P3D4 polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 151P3D4.A typical embodiment of this invention provides methods for monitoring151P3D4 gene products in a tissue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 151P3D4such as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of151P3D4 as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 151P3D4 in a human subject wherein the compositioncomprises a carrier suitable for human use and a human unit dose of oneor more than one agent that inhibits the production or function of151P3D4. Preferably, the carrier is a uniquely human carrier. In anotheraspect of the invention, the agent is a moiety that is immunoreactivewith 151P3D4 protein. Non-limiting examples of such moieties include,but are not limited to, antibodies (such as single chain, monoclonal,polyclonal, humanized, chimeric, or human antibodies), functionalequivalents thereof (whether naturally occurring or synthetic), andcombinations thereof. The antibodies can be conjugated to a diagnosticor therapeutic moiety. In another aspect, the agent is a small moleculeas defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to 151P3D4 and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with 151P3D4 as described above.The one or more than one nucleic acid molecule may also be, or encodes,a molecule that inhibits production of 151P3D4. Non-limiting examples ofsuch molecules include, but are not limited to, those complementary to anucleotide sequence essential for production of 151P3D4 (e.g. antisensesequences or molecules that form a triple helix with a nucleotide doublehelix essential for 151P3D4 production) or a ribozyme effective to lyse151P3D4 mRNA.

Another embodiment of the invention is antibody epitopes which comprisea peptide regions, or an oligonucleotide encoding the peptide region,that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equalto 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Beta-turn profile of FIG. 9.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 151P3D4 SSH sequence of 417 nucleotides.

FIG. 2. The cDNA and amino acid sequence of 151P3D4 v.1 clone 1-placenta(also called “151P3D4 v.1” or “151P3D4 variant 1”) is shown in FIG. 2A.The start methionine is underlined. The open reading frame extends fromnucleic acid 316-1380 including the stop codon. The cDNA and amino acidsequence of 151P3D4 variant 2 (also called “151P3D4 v.2”) is shown inFIG. 2B. The codon for the start methionine is underlined. The openreading frame extends from nucleic acid 1-2166 including the stop codon.The cDNA and amino acid sequence of 151P3D4 variant 3 (also called“151P3D4 v.3”) is shown in FIG. 2C. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 316-1380including the stop codon. The cDNA and amino acid sequence of 151P3D4variant 4 (also called “151P3D4 v.4”) is shown in FIG. 2D. The codon forthe start methionine is underlined. The open reading frame extends fromnucleic acid 316-1380 including the stop codon. The cDNA and amino acidsequence of 151P3D4 variant 5 (also called “151P3D4 v.5”) is shown inFIG. 2E. The codon for the start methionine is underlined. The openreading frame extends from nucleic acid 316-1380 including the stopcodon. The cDNA and amino acid sequence of 151P3D4 variant 6 (alsocalled “151P3D4 v.6”) is shown in FIG. 2F. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 316-1380 including the stop codon. The cDNA and amino acid sequenceof 151P3D4 variant 7 (also called “151P3D4 v.7”) is shown in FIG. 2G.The codon for the start methionine is underlined. The open reading frameextends from nucleic acid 316-1380 including the stop codon. The cDNAand amino acid sequence of 151P3D4 variant 8 (also called “151P3D4 v.8”)is shown in FIG. 2H. The codon for the start methionine is underlined.The open reading frame extends from nucleic acid 316-1380 including thestop codon. The cDNA and amino acid sequence of 151P3D4 variant 9 (alsocalled “151P3D4 v.9”) is shown in FIG. 2I. The codon for the startmethionine is underlined. The open reading frame extends from nucleicacid 316-1380 including the stop codon. The cDNA and amino acid sequenceof 151P3D4 variant 10 (also called “151P3D4 v.10”) is shown in FIG. 2J.The codon for the start methionine is underlined. The open reading frameextends from nucleic acid 316-1380 including the stop codon. The cDNAand amino acid sequence of 151P3D4 variant 11 (also called “151P3D4v.11”) is shown in FIG. 2K. The codon for the start methionine isunderlined. The open reading frame extends from nucleic acid 316-1380including the stop codon. As used herein, a reference to 151P3D4includes all variants thereof, including those shown in FIGS. 10 and 12.

FIG. 3. Amino acid sequence of 151P3D4 v.1 is shown in FIG. 3A; it has354 amino acids. The amino acid sequence of 151P3D4 v.2 is shown in FIG.3B; it has 721 amino acids. As used herein, a reference to 151P3D4includes all variants thereof, including those shown in FIGS. 11 and 12.

FIG. 4. The nucleic acid sequence alignment of 151P3D4 v.1 with the mRNAfor human cartilage link protein is shown in FIG. 4A. The amino acidsequence alignments of 151P3D4 v.1 with human cartilage link protein(4B), mouse cartilage link protein (4C), 151P3D4 v.2 (4D), hypotheticalprotein XP_(—)094318 (4E), bovine cartilage link protein (4F), and ratcartilage link protein (4G) are shown in FIGS. 4B-4G. The amino acidsequence alignments of 151P3D4 v.2 with human cartilage link protein isshown in FIG. 4H. The clustal alignment of 151P3D4 v.1 and 151P3D4 v.2is shown in FIG. 4I.

FIG. 5. Hydrophilicity amino acid profile of A) 151P3D4 v.1 and B)151P3D4 v.2, determined by computer algorithm sequence analysis usingthe method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale Internetwebsite (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecularbiology server.

FIG. 6. Hydropathicity amino acid profile of A) 151P3D4 v.1 and B)151P3D4 v.2, determined by computer algorithm sequence analysis usingthe method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J.Mol. Biol. 157:105-132) accessed on the ProtScale Internet website(expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 7. Percent accessible residues amino acid profile of A) 151P3D4 v.1and B) 151P3D4 v.2, determined by computer algorithm sequence analysisusing the method of Janin (Janin J., 1979 Nature 277:491-492) accessedon the ProtScale Internet website (expasy.ch/cgi-bin/protscale.pl)through the ExPasy molecular biology server.

FIG. 8. Average flexibility amino acid profile of A) 151P3D4 v.1 and B)151P3D4 v.2, determined by computer algorithm sequence analysis usingthe method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on theProtScale Internet website (expasy.ch/cgi-bin/protscale.pl) through theExPasy molecular biology server.

FIG. 9. Beta-turn amino acid profile of A) 151P3D4 v.1 and B) 151P3D4v.2, determined by computer algorithm sequence analysis using the methodof Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering1:289-294) accessed on the ProtScale Internet website(expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biologyserver.

FIG. 10. Schematic display of nucleotide variants of 151P3D4. Schematicalignment of Single Nucleotide Polymorphism (SNP) variants of 151P3D4.Variants 151P3D4 v.3 through v.11 are variants with single nucleotidedifferences. Though these SNP variants are shown separately, they couldalso occur in any combinations and in any one of the transcript variantsthat contains the base pairs. Numbers correspond to those of 151P3D4v.1. The black boxes show the same sequence as 151P3D4 v.1. SNPs areindicated above the boxes.

FIG. 11. Schematic alignment of protein variants of 151P3D4. Nucleotidevariants 151P3D4 v.2 through v.9 in FIG. 10 code for the same protein as151P3D4 v.1. Variants 151P3D4 v.2 codes for a protein that shares 321 aawith 151P3D4 v.1. Boxes with the same fill pattern represent the samesequence. Numbers in “( )” underneath the boxes correspond to 151P3D4v.1.

FIG. 12. Schematic alignment of transcript variants of 151P3D4. Variant151P3D4 v.2 is an alternative transcript, which shares the last threeexons with 151P3D4 v.1. The first two exons of 151P3D4 v.1 are locatedin the sixth intron (between exons 6 and 7) of 151P3D4 v.2. Numbers in“( )” underneath the boxes correspond to those of 151P3D4 v.2. Boxeswith the same fill pattern represent the same sequence.

FIG. 13. Secondary structure prediction for 151P3D4 protein variants.The secondary structure of 151P3D4 protein variants 1 and 2 (FIGS. A(SEQ ID NO. 66) and B (SEQ ID NO. 67), respectively) were predictedusing the HNN—Hierarchical Neural Network method (Guermeur, 1997,located on the World Wide Web at:pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed fromthe ExPasy molecular biology server from Internet website(expasy.ch/tools/). This method predicts the presence and location ofalpha helices, extended strands, and random coils from the primaryprotein sequence. The percent of the protein in a given secondarystructure is also listed.

FIG. 14. Expression of 151P3D4 by RT-PCR. First strand cDNA was preparedfrom vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas,colon and stomach), bladder cancer pool, kidney cancer pool, coloncancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,and cancer metastasis pool. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primers to151P3D4, was performed at 26 and 30 cycles of amplification. Resultsshow strong expression of 151P3D4 in ovary cancer pool. Expression of151P3D4 was also detected in bladder cancer pool, kidney cancer pool,colon cancer pool, lung cancer pool, breast cancer pool, cancermetastasis pool, vital pool 2, but not in vital pool 1.

FIG. 15. Expression of 151P3D4 in normal tissues. Two multiple tissuenorthern blots (Clontech) both with 2 μg of mRNA/lane were probed withthe 151P3D4 sequence. Size standards in kilobases (kb) are indicated onthe side. Results show expression of 151P3D4 in small intestine andplacenta. Lower level expression was also detected in heart and colon,but not in the other normal tissues tested.

FIG. 16. Expression of 151P3D4 in bladder cancer patient tissues. RNAwas extracted from normal bladder (NB), bladder cancer cell lines (CL:UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) and normaladjacent tissue (NAT). Northern blots with 10 μg of total RNA wereprobed with the 151P3D4 SSH sequence. Size standards in kilobases areindicated on the side. Results show expression of 151P3D4 in patientbladder cancer tissues, and in UM-UC-3 bladder cancer cell lines, butnot in normal bladder nor in the other bladder cancer cell lines tested.

FIG. 17. Expression of 151P3D4 in kidney cancer patient tissues. RNA wasextracted from kidney cancer cell lines (CL: 769-P, A498, SW839), normalkidney (NK), kidney cancer patient tumors (T) and their normal adjacenttissues (NAT). Northern blots with 10 μg of total RNA were probed withthe 151P3D4 SSH sequence. Size standards in kilobases are on the side.Results show expression of 151P3D4 in patient kidney tumor tissues, butnot in normal kidney, nor in the cell lines tested.

FIG. 18. Expression of 151P3D4 in ovary cancer patient tissues. RNA wasextracted from ovary and cervical cancer cell lines (CL), normal ovary(N), and ovary cancer patient tumor (T). Northern blots with 10 μg oftotal RNA were probed with the 151P3D4 SSH sequence. Size standards inkilobases are on the side. Results show strong expression of 151P3D4 inpatient ovary cancer tissues, but not in normal ovary nor in the ovaryand cervical cancer cell lines.

FIG. 19. Expression of 151P3D4 in stomach and uterus human cancerspecimens. Expression of 151P3D4 was assayed in a panel of human stomachand uterus cancers (T) and their respective matched normal tissues (N)on RNA dot blots. 151P3D4 expression was seen in both stomach and uteruscancers.

FIG. 20. 151P3D4 expression in 293T cells following transfection. 293Tcells were transfected with either 151P3D4 .pcDNA3.1/mychis orpcDNA3.1/mychis vector control. Forty hours later, cell lysates werecollected. Samples were run on an SDS-PAGE acrylamide gel, blotted andstained with anti-his antibody. The blot was developed using the ECLchemiluminescence kit and visualized by autoradiography. Results showexpression of 151P3D4 from the 151P3D4 .pcDNA3.1/mychis mammalianexpression construct in the lysates of 151P3D4 .pcDNA3.1/mychistransfected cells, but not from the control pcDNA3.1/mychis vector.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C₁-C₂ disease under the Whitmore-Jewett system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 151P3D4 (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence 151P3D4. In addition, the phrase includesqualitative changes in the glycosylation of the native proteins,involving a change in the nature and proportions of the variouscarbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a151P3D4-related protein). For example an analog of a 151P3D4 protein canbe specifically bound by an antibody or T cell that specifically bindsto 151P3D4.

The term “antibody” is used in the broadest sense. Therefore an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-151P3D4antibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-151P3D4 antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-151P3D4 antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to maytansinoids, yttrium,bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol,ethidium bromide, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicine, dihydroxy anthracin dione, actinomycin,diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin Achain, modeccin A chain, alpha-sarcin, gelonin, mitogellin,retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,sapaonaria officinalis inhibitor, and glucocorticoid and otherchemotherapeutic agents, as well as radioisotopes such as At²¹¹, I¹³¹,I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes ofLu. Antibodies may also be conjugated to an anti-cancer pro-drugactivating enzyme capable of converting the pro-drug to its active form.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 151P3D4 genes orthat encode polypeptides other than 151P3D4 gene product or fragmentsthereof. A skilled artisan can readily employ nucleic acid isolationprocedures to obtain an isolated 151P3D4 polynucleotide. A protein issaid to be “isolated,” for example, when physical, mechanical orchemical methods are employed to remove the 151P3D4 proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 151P3D4 protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage T×N×M+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A “motif”, as in biological motif of a 151P3D4-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the pattern of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class 1 molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. In another embodiment, for example, the primary anchorresidues of a peptide that will bind an HLA class II molecule are spacedrelative to each other, rather than to the termini of a peptide, wherethe peptide is generally of at least 9 amino acids in length. Theprimary anchor positions for each motif and supermotif are set forth inTable IV. For example, analog peptides can be created by altering thepresence or absence of particular residues in the primary and/orsecondary anchor positions shown in Table IV. Such analogs are used tomodulate the binding affinity and/or population coverage of a peptidecomprising a particular HLA motif or supermotif.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that hasbeen subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind orinteract with 151P3D4, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit 151P3D4 protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, 151P3D4 protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium. citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C. “Moderately stringent conditions” are described by, but not limitedto, those in Sambrook et al., Molecular Cloning: A Laboratory Manual,New York: Cold Spring Harbor Press, 1989, and include the use of washingsolution and hybridization conditions (e.g., temperature, ionic strengthand % SDS) less stringent than those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles.

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 151P3D4 protein shown in FIG. 2 or FIG. 3.An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “151P3D4-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 151P3D4 proteins orfragments thereof, as well as fusion proteins of a 151P3D4 protein and aheterologous polypeptide are also included. Such 151P3D4 proteins arecollectively referred to as the 151P3D4-related proteins, the proteinsof the invention, or 151P3D4. The term “151P3D4-related protein” refersto a polypeptide fragment or a 151P3D4 protein sequence of 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, ormore than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65,70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275,300, 325, 350, or 354 or more amino acids.

151P3D4 Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 151P3D4 gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 151P3D4-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a 151P3D4 gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa 151P3D4 gene, mRNA, or to a 151P3D4 encoding polynucleotide(collectively, “151P3D4 polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 151P3D4 polynucleotide include: a 151P3D4polynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 151P3D4 as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 151P3D4 nucleotides comprise, without limitation:

(I) a polynucleotide comprising, consisting essentially of, orconsisting of a sequence as shown in FIG. 2, wherein T can also be U;

(II) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2A, from nucleotide residuenumber 316 through nucleotide residue number 1380, including the stopcodon, wherein T can also be U;

(III) a polynucleotide comprising, consisting essentially of, orconsisting of the sequence as shown in FIG. 2B, from nucleotide residuenumber 1 through nucleotide residue number 2166, including the stopcodon, wherein T can also be U;

(IV) a polynucleotide comprising, consisting essentially of, orconsisting of the sequences as shown in FIGS. 2C-2K, from nucleotideresidue number 316 through nucleotide residue number 1380, including thea stop codon, wherein T can also be U;

(V) a polynucleotide that encodes a 151P3D4-related protein that is atleast 90% homologous to an entire amino acid sequence shown in FIG.2A-K;

(VI) a polynucleotide that encodes a 151P3D4-related protein that is atleast 90% identical to an entire amino acid sequence shown in FIG. 2A-K;

(VII) a polynucleotide that encodes at least one peptide set forth inTables V-XVIII and XXII-LI;

(VIII) a polynucleotide that encodes a peptide region of at least 5amino acids of a peptide of FIG. 3A in any whole number increment up to354 that includes an amino acid position having a value greater than 0.5in the Hydrophilicity profile of FIG. 5A; or of FIG. 3B in any wholenumber increment up to 721 that includes an amino acid position having avalue greater than 0.5 in the Hydrophilicity profile of FIG. 5B;

(XIX) a polynucleotide that encodes a peptide region of at least 5 aminoacids of a peptide of FIG. 3A in any whole number increment up to 354that includes an amino acid position having a value less than 0.5 in theHydropathicity profile of FIG. 6A; or of FIG. 3B in any whole numberincrement up to 721 that includes an amino acid position having a valueless than 0.5 in the Hydropathicity profile of FIG. 6B;

(X) a polynucleotide that encodes a peptide region of at least 5 aminoacids of a peptide of FIG. 3A in any whole number increment up to 354that includes an amino acid position having a value greater than 0.5 inthe Percent Accessible Residues profile of FIG. 7A; or of FIG. 3B in anywhole number increment up to 721 that includes an amino acid positionhaving a value greater than 0.5 in the Percent Accessible Residuesprofile of FIG. 7B;

(XII) a polynucleotide that encodes a peptide region of at least 5 aminoacids of a peptide of FIG. 3A in any whole number increment up to 354that includes an amino acid position having a value greater than 0.5 inthe Average Flexibility profile of FIG. 8A; or of FIG. 3B in any wholenumber increment up to 721 that includes an amino acid position having avalue greater than 0.5 in the Average Flexibility profile of FIG. 8B;

(XIII) a polynucleotide that encodes a peptide region of at least 5amino acids of a peptide of FIG. 3A in any whole number increment up to354 that includes an amino acid position having a value greater than 0.5in the Beta-turn profile of FIG. 9A; or of FIG. 3B in any whole numberincrement up to 721 that includes an amino acid position having a valuegreater than 0.5 in the Beta-turn profile of FIG. 9B;

(XIV) a polynucleotide that is fully complementary to a polynucleotideof any one of (I)-(XIII).

(XV) a peptide that is encoded by any of (I)-(XIV); and

(XVI) a polynucleotide of any of (I)-(XIV) or peptide of (XV) togetherwith a pharmaceutical excipient and/or in a human unit dose form.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 151P3D4polynucleotides that encode specific portions of 151P3D4 mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, or 354or more contiguous amino acids of 151P3D4.

(b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375,400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, or 721or more contiguous amino acids of 151P3D4 variant 2.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 151P3D4protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 151P3D4 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 20 to about amino acid30 of the 151P3D4 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 30 to about amino acid 40 of the 151P3D4protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 40 to about amino acid 50 of the 151P3D4 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 50 to about amino acid60 of the 151P3D4 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 60 to about amino acid 70 of the 151P3D4protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 70 to about amino acid 80 of the 151P3D4 protein shown in FIG. 2 orFIG. 3, polynucleotides encoding about amino acid 80 to about amino acid90 of the 151P3D4 protein shown in FIG. 2 or FIG. 3, polynucleotidesencoding about amino acid 90 to about amino acid 100 of the 151P3D4protein shown in FIG. 2 or FIG. 3, in increments of about 10 aminoacids, ending at the carboxyl terminal amino acid set forth in FIG. 2 orFIG. 3. Accordingly polynucleotides encoding portions of the amino acidsequence (of about 10 amino acids), of amino acids 100 through thecarboxyl terminal amino acid of the 151P3D4 protein are embodiments ofthe invention. Wherein it is understood that each particular amino acidposition discloses that position plus or minus five amino acid residues.

Polynucleotides encoding relatively long portions of a 151P3D4 proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 151P3D4 protein “or variant”shown in FIG. 2 or FIG. 3 can be generated by a variety of techniqueswell known in the art. These polynucleotide fragments can include anyportion of the 151P3D4 sequence as shown in FIG. 2.

One embodiment of the invention comprises an HLA peptide, that occurs atleast twice in Tables V-XVIII and XXII to LI collectively, or anoligonucleotide that encodes the HLA peptide. Another embodiment of theinvention comprises an HLA peptide that occurs at least once in TablesV-XVIII and at least once in tables XXII to LI, or an oligonucleotidethat encodes the HLA peptide. In another embodiment of the invention,typical polynucleotide fragments can encode one or more of the 151P3D4protein or variant N-glycosylation sites, cAMP and cGMP-dependentprotein kinase phosphorylation sites, casein kinase II phosphorylationsites or N-myristoylation site and amidation sites.

Uses of 151P3D4 Polynucleotides

Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 151P3D4 gene maps to the chromosomallocation set forth in the Example entitled “Chromosomal Mapping of151P3D4.” For example, because the 151P3D4 gene maps to this chromosome,polynucleotides that encode different regions of the 151P3D4 proteinsare used to characterize cytogenetic abnormalities of this chromosomallocale, such as abnormalities that are identified as being associatedwith various cancers. In certain genes, a variety of chromosomalabnormalities including rearrangements have been identified as frequentcytogenetic abnormalities in a number of different cancers (see e.g.Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al.,Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23):9158-9162 (1988)). Thus, polynucleotides encoding specific regions ofthe 151P3D4 proteins provide new tools that can be used to delineate,with greater precision than previously possible, cytogeneticabnormalities in the chromosomal region that encodes 151P3D4 that maycontribute to the malignant phenotype. In this context, thesepolynucleotides satisfy a need in the art for expanding the sensitivityof chromosomal screening in order to identify more subtle and lesscommon chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet.Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 151P3D4 was shown to be highly expressed in bladder andother cancers, 151P3D4 polynucleotides are used in methods assessing thestatus of 151P3D4 gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 151P3D4proteins are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 151P3D4 gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378(1999), both of which utilize polynucleotides encoding specific regionsof a protein to examine these regions within the protein.

Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 151P3D4. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives, that specifically bind DNA or RNA in abase pair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 151P3D4 polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,151P3D4. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 151P3D4 antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990).Additional 151P3D4 antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

The 151P3D4 antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a151P3D4 genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 151P3D4 mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 151P3D4 antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 151P3D4 mRNA. Optionally, 151P3D4antisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 151P3D4. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 151P3D4 expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet. 12: 510-515 (1996).

Primers and Primer Pairs

Further specific embodiments of this nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 151P3D4 polynucleotide in a sample and as ameans for detecting a cell expressing a 151P3D4 protein.

Examples of such probes include polypeptides comprising all or part ofthe human 151P3D4 cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 151P3D4 mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 151P3D4 mRNA.

The 151P3D4 polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the 151P3D4 gene(s), mRNA(s),or fragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of 151P3D4 polypeptides; as tools formodulating or inhibiting the expression of the 151P3D4 gene(s) and/ortranslation of the 151P3D4 transcript(s); and as therapeutic agents.

The present invention includes the use of any probe as described hereinto identify and isolate a 151P3D4 or 151P3D4 related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

Isolation of 151P3D4-Encoding Nucleic Acid Molecules

The 151P3D4 cDNA sequences described herein enable the isolation ofother polynucleotides encoding 151P3D4 gene product(s), as well as theisolation of polynucleotides encoding 151P3D4 gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a151P3D4 gene product as well as polynucleotides that encode analogs of151P3D4-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 151P3D4 gene are wellknown (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 151P3D4gene cDNAs can be identified by probing with a labeled 151P3D4 cDNA or afragment thereof. For example, in one embodiment, a 151P3D4 cDNA (e.g.,FIG. 2) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full-length cDNAs corresponding to a 151P3D4gene. A 151P3D4 gene itself can be isolated by screening genomic DNAlibraries, bacterial artificial chromosome libraries (BACs), yeastartificial chromosome libraries (YACs), and the like, with 151P3D4 DNAprobes or primers.

Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 151P3D4 polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart, and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 151P3D4 polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPr1, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of 151P3D4or a fragment, analog or homolog thereof can be used to generate 151P3D4proteins or fragments thereof using any number of host-vector systemsroutinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of151P3D4 proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 151P3D4 can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPr1. The host-vector systems of the invention are useful forthe production of a 151P3D4 protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 151P3D4 and 151P3D4 mutations or analogs.

Recombinant human 151P3D4 protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 151P3D4-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 151P3D4 or fragment,analog or homolog thereof, a 151P3D4-related protein is expressed in the293T cells, and the recombinant 151P3D4 protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-151P3D4 antibodies). In another embodiment, a 151P3D4 codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 andrat-1 in order to establish 151P3D4 expressing cell lines. Various otherexpression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to a151P3D4 coding sequence can be used for the generation of a secretedform of recombinant 151P3D4 protein.

As discussed herein, redundancy in the genetic code permits variation in151P3D4 gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the Internet such as at URLdna.affrc.go.jp/˜nakamura/codon.html.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell. Biol., 9:5073-5080(1989) Skilled artisans understand that the general rule that eukaryoticribosomes initiate translation exclusively at the 5′ proximal AUG codonis abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7):2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

151P3D4-Related Proteins

Another aspect of the present invention provides 151P3D4-relatedproteins. Specific embodiments of 151P3D4 proteins comprise apolypeptide having all or part of the amino acid sequence of human151P3D4 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of151P3D4 proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 151P3D4 shown in FIG. 2or FIG. 3.

In general, naturally occurring allelic variants of human 151P3D4 sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of a 151P3D4 protein containconservative amino acid substitutions within the 151P3D4 sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 151P3D4. One class of 151P3D4allelic variants are proteins that share a high degree of homology withat least a small region of a particular 151P3D4 amino acid sequence, butfurther contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry”2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 151P3D4 proteins such aspolypeptides having amino acid insertions, deletions and substitutions.151P3D4 variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 151P3D4 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia,J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yieldadequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 151P3D4 variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 151P3D4 protein having an amino acid sequence of FIG.3. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to a 151P3D4 variant also specificallybinds to a 151P3D4 protein having an amino acid sequence set forth inFIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG.3, when it no longer contains any epitope capable of being recognized byan antibody or T cell that specifically binds to the starting 151P3D4protein. Those skilled in the art understand that antibodies thatrecognize proteins bind to epitopes of varying size, and a grouping ofthe order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol. 2000 165(12): 6949-6955; Hebbes et al.,Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985)135(4):2598-608.

Other classes of 151P3D4-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with an amino acid sequence of FIG. 3, ora fragment thereof. Another specific class of 151P3D4 protein variantsor analogs comprise one or more of the 151P3D4 biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 151P3D4 fragments (nucleic or aminoacid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a151P3D4 protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a151P3D4 protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of a 151P3D4 protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of a 151P3D4protein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of a 151P3D4 protein shown in FIG.2 or FIG. 3, polypeptides consisting of about amino acid 30 to aboutamino acid 40 of a 151P3D4 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 151P3D4 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a 151P3D4 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a 151P3D4 protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa 151P3D4 protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a 151P3D4 protein shown inFIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a 151P3D4 protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a 151P3D4 amino acid sequence. Moreover,polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.)to about amino acid 20, (or 130, or 140 or 150 etc.) of a 151P3D4protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. Itis to be appreciated that the starting and stopping positions in thisparagraph refer to the specified position as well as that position plusor minus 5 residues.

151P3D4-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 151P3D4-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofa 151P3D4 protein (or variants, homologs or analogs thereof).

Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 151P3D4 polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within a 151P3D4 polypeptidesequence set forth in FIG. 2 or FIG. 3. Various motifs are known in theart, and a protein can be evaluated for the presence of such motifs by anumber of publicly available Internet sites (see, e.g., URL addresses:pfam.wustl.edu/;searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html;psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk; ebi.ac.uk/interpro/scan.html;expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University,brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,bimas.dcrt.nih.gov/).

Motif bearing subsequences of all 151P3D4 variant proteins are set forthand identified in Tables V-XVIII and XXII-LII.

Table XIX sets forth several frequently occurring motifs based on pfamsearches (see URL address pfam.wustl.edu/). The columns of Table XIXlist (1) motif name abbreviation, (2) percent identity found amongst thedifferent member of the motif family, (3) motif name or description and(4) most common function; location information is included if the motifis relevant for location.

Polypeptides comprising one or more of the 151P3D4 motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 151P3D4 motifsdiscussed above are associated with growth dysregulation and because151P3D4 is overexpressed in certain cancers (See, e.g., Table I). Caseinkinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C,for example, are enzymes known to be associated with the development ofthe malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995);Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterzielet al., Oncogene 18(46): 6322-6329 (1999) and O′Brian, Oncol. Rep. 5(2):305-309 (1998)). Moreover, both glycosylation and myristoylation areprotein modifications also associated with cancer and cancer progression(see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999);Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation isanother protein modification also associated with cancer and cancerprogression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr.(13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables V-XVIII and XXII-LI.CTL epitopes can be determined using specific algorithms to identifypeptides within a 151P3D4 protein that are capable of optimally bindingto specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, BrownUniversity, Internet URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URLbimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides thathave sufficient binding affinity for HLA molecules and which arecorrelated with being immunogenic epitopes, are well known in the art,and are carried out without undue experimentation. In addition,processes for identifying peptides that are immunogenic epitopes, arewell known in the art, and are carried out without undue experimentationeither in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, one can substitute out a deleterious residue infavor of any other residue, such as a preferred residue as defined inTable IV; substitute a less-preferred residue with a preferred residueas defined in Table IV; or substitute an originally-occurring preferredresidue with another preferred residue as defined in Table IV.Substitutions can occur at primary anchor positions or at otherpositions in a peptide; see, e.g., Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 97/33602 to Chesnut et al.; Sette,Immunogenetics 1999 50(3-4): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149: 3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprisingcombinations of the different motifs set forth in Table XX, and/or, oneor more of the predicted CTL epitopes of Tables V-XVII and XXII-XLVII,and/or, one or more of the predicted HTL epitopes of Tables XLVIII-LI,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typicallythe number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

151P3D4-related proteins are embodied in many forms, preferably inisolated form. A purified 151P3D4 protein molecule will be substantiallyfree of other proteins or molecules that impair the binding of 151P3D4to antibody, T cell or other ligand. The nature and degree of isolationand purification will depend on the intended use. Embodiments of a151P3D4-related proteins include purified 151P3D4-related proteins andfunctional, soluble 151P3D4-related proteins. In one embodiment, afunctional, soluble 151P3D4 protein or fragment thereof retains theability to be bound by antibody, T cell or other ligand.

The invention also provides 151P3D4 proteins comprising biologicallyactive fragments of a 151P3D4 amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the starting 151P3D4protein, such as the ability to elicit the generation of antibodies thatspecifically bind an epitope associated with the starting 151P3D4protein; to be bound by such antibodies; to elicit the activation of HTLor CTL; and/or, to be recognized by HTL or CTL that also specificallybind to the starting protein.

151P3D4-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or on the basis of immunogenicity. Fragmentsthat contain such structures are particularly useful in generatingsubunit-specific anti-151P3D4 antibodies, or T cells or in identifyingcellular factors that bind to 151P3D4. For example, hydrophilicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%) Accessible Residues profiles can be generated,and immunogenic peptide fragments identified, using the method of JaninJ., 1979, Nature 277:491-492. Average Flexibility profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. ProteinRes. 32:242-255. Beta-turn profiles can be generated, and immunogenicpeptide fragments identified, using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identifypeptides within a 151P3D4 protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site at WorldWide Web URL syfpeithi.bmi-heidelberg.com/; the listings in TableIV(A)-(E); Epimatrix™ and Epimer™, Brown University, Internet URL(brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URLbimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 151P3D4that are presented in the context of human MHC Class I molecules, e.g.,HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., TablesV-XVIII, XXII-LI). Specifically, the complete amino acid sequence of the151P3D4 protein and relevant portions of other variants, i.e., for HLAClass I predictions 9 flanking residues on either side of a pointmutation, and for HLA Class II predictions 14 flanking residues oneither side of a point mutation, were entered into the HLA Peptide MotifSearch algorithm found in the Bioinformatics and Molecular AnalysisSection (BIMAS) web site listed above; in addition to the siteSYFPEITHI, at URL syfpeithi.bmi-heidelberg.com/.

The HLA peptide motif search algorithm was developed by Dr. Ken Parkerbased on binding of specific peptide sequences in the groove of HLAClass I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker etal., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol.152:163-75 (1994)). This algorithm allows location and ranking of 8-mer,9-mer, and 10-mer peptides from a complete protein sequence forpredicted binding to HLA-A2 as well as numerous other HLA Class Imolecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers.For example, for Class I HLA-A2, the epitopes preferably contain aleucine (L) or methionine (M) at position 2 and a valine (V) or leucine(L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7(1992)). Selected results of 151P3D4 predicted binding peptides areshown in Tables V-XVIII and XXII-LI herein. In Tables V-XVIII andXXII-XLVII, selected candidates, 9-mers and 10-mers, for each familymember are shown along with their location, the amino acid sequence ofeach specific peptide, and an estimated binding score. In TablesXLVIII-LI, selected candidates, 15-mers, for each family member areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score are predicted to be the most tightly bound to HLA Class Ion the cell surface for the greatest period of time and thus representthe best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using World Wide Web site URLsyfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) are to be“applied” to a 151P3D4 protein in accordance with the invention. As usedin this context “applied” means that a 151P3D4 protein is evaluated,e.g., visually or by computer-based patterns finding methods, asappreciated by those of skill in the relevant art. Every subsequence ofa 151P3D4 protein of 8, 9, 10, or 11 amino acid residues that bears anHLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

Expression of 151P3D4-Related Proteins

In an embodiment described in the examples that follow, 151P3D4 can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 151P3D4 with a C-terminal 6× His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 151P3D4 protein intransfected cells. The secreted HIS-tagged 151P3D4 in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

Modifications of 151P3D4-Related Proteins

Modifications of 151P3D4-related proteins such as covalent modificationsare included within the scope of this invention. One type of covalentmodification includes reacting targeted amino acid residues of a 151P3D4polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues of a151P3D4 protein. Another type of covalent modification of a 151P3D4polypeptide included within the scope of this invention comprisesaltering the native glycosylation pattern of a protein of the invention.Another type of covalent modification of 151P3D4 comprises linking a151P3D4 polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 151P3D4-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 151P3D4 fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a151P3D4 sequence (amino or nucleic acid) such that a molecule is createdthat is not, through its length, directly homologous to the amino ornucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimericmolecule can comprise multiples of the same subsequence of 151P3D4. Achimeric molecule can comprise a fusion of a 151P3D4-related proteinwith a polyhistidine epitope tag, which provides an epitope to whichimmobilized nickel can selectively bind, with cytokines or with growthfactors. The epitope tag is generally placed at the amino- orcarboxyl-terminus of a 151P3D4 protein. In an alternative embodiment,the chimeric molecule can comprise a fusion of a 151P3D4-related proteinwith an immunoglobulin or a particular region of an immunoglobulin. Fora bivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a 151P3D4polypeptide in place of at least one variable region within an Igmolecule. In a preferred embodiment, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CHI, CH2 and CH3 regions of anIgGI molecule. For the production of immunoglobulin fusions see, e.g.,U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

Uses of 151P3D4-Related Proteins

The proteins of the invention have a number of different specific uses.As 151P3D4 is highly expressed in prostate and other cancers,151P3D4-related proteins are used in methods that assess the status of151P3D4 gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 151P3D4 protein are used to assess the presence ofperturbations (such as deletions, insertions, point mutations etc.) inthose regions (such as regions containing one or more motifs). Exemplaryassays utilize antibodies or T cells targeting 151P3D4-related proteinscomprising the amino acid residues of one or more of the biologicalmotifs contained within a 151P3D4 polypeptide sequence in order toevaluate the characteristics of this region in normal versus canceroustissues or to elicit an immune response to the epitope. Alternatively,151P3D4-related proteins that contain the amino acid residues of one ormore of the biological motifs in a 151P3D4 protein are used to screenfor factors that interact with that region of 151P3D4.

151P3D4 protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a151P3D4 protein), for identifying agents or cellular factors that bindto 151P3D4 or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 151P3D4 genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 151P3D4 gene product.Antibodies raised against a 151P3D4 protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 151P3D4protein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 151P3D4-related nucleic acids or proteins are also used ingenerating HTL or CTL responses.

Various immunological assays useful for the detection of 151P3D4proteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 151P3D4-expressingcells (e.g., in radioscintigraphic imaging methods). 151P3D4 proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

151P3D4 Antibodies

Another aspect of the invention provides antibodies that bind to151P3D4-related proteins. Preferred antibodies specifically bind to a151P3D4-related protein and do not bind (or bind weakly) to peptides orproteins that are not 151P3D4-related proteins. For example, antibodiesthat bind 151P3D4 can bind 151P3D4-related proteins such as the homologsor analogs thereof.

151P3D4 antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of other cancers, to the extent 151P3D4 isalso expressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of151P3D4 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 151P3D4 and mutant 151P3D4-relatedproteins. Such assays can comprise one or more 151P3D4 antibodiescapable of recognizing and binding a 151P3D4-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 151P3D4 are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 151P3D4 antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 151P3D4 expressingcancers such as prostate cancer.

151P3D4 antibodies are also used in methods for purifying a151P3D4-related protein and for isolating 151P3D4 homologues and relatedmolecules. For example, a method of purifying a 151P3D4-related proteincomprises incubating a 151P3D4 antibody, which has been coupled to asolid matrix, with a lysate or other solution containing a151P3D4-related protein under conditions that permit the 151P3D4antibody to bind to the 151P3D4-related protein; washing the solidmatrix to eliminate impurities; and eluting the 151P3D4-related proteinfrom the coupled antibody. Other uses of 151P3D4 antibodies inaccordance with the invention include generating anti-idiotypicantibodies that mimic a 151P3D4 protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 151P3D4-related protein, peptide, or fragment, inisolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSHPress, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold SpringHarbor Press, NY (1989)). In addition, fusion proteins of 151P3D4 canalso be used, such as a 151P3D4 GST-fusion protein. In a particularembodiment, a GST fusion protein comprising all or most of the aminoacid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogento generate appropriate antibodies. In another embodiment, a151P3D4-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 151P3D4-related protein or 151P3D4 expressingcells) to generate an immune response to the encoded immunogen (forreview, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of a 151P3D4 protein as shown in FIG. 2 or FIG.3 can be analyzed to select specific regions of the 151P3D4 protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a 151P3D4 amino acid sequence are used to identifyhydrophilic regions in the 151P3D4 structure. Regions of a 151P3D4protein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R. F., 1982, J.Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can begenerated using the method of Janin J., 1979, Nature 277:491-492.Average Flexibility profiles can be generated using the method ofBhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, eachregion identified by any of these programs or methods is within thescope of the present invention. Methods for the generation of 151P3D4antibodies are further illustrated by way of the examples providedherein. Methods for preparing a protein or polypeptide for use as animmunogen are well known in the art. Also well known in the art aremethods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 151P3D4 immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art. During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

151P3D4 monoclonal antibodies can be produced by various means wellknown in the art. For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 151P3D4-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof a 151P3D4 protein can also be produced in the context of chimeric orcomplementarity determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human 151P3D4 antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 151P3D4 monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human151P3D4 monoclonal antibodies can also be produced using transgenic miceengineered to contain human immunoglobulin gene loci as described in PCTPatent Application WO98/24893, Kucherlapati and Jakobovits et al.,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued 19 Dec. 2000;6,150,584 issued 12 Nov. 2000; and, 6,114,598 issued 5 Sep. 2000). Thismethod avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 151P3D4 antibodies with a 151P3D4-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,151P3D4-related proteins, 151P3D4-expressing cells or extracts thereof.A 151P3D4 antibody or fragment thereof can be labeled with a detectablemarker or conjugated to a second molecule. Suitable detectable markersinclude, but are not limited to, a radioisotope, a fluorescent compound,a bioluminescent compound, chemiluminescent compound, a metal chelatoror an enzyme. Further, bi-specific antibodies specific for two or more151P3D4 epitopes are generated using methods generally known in the art.Homodimeric antibodies can also be generated by cross-linking techniquesknown in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

151P3D4 Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web atURL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al.,Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995;Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., HumanImmunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999November; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D.C., J. Mol.Biol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol.8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

151P3D4 Transgenic Animals

Nucleic acids that encode a 151P3D4-related protein can also be used togenerate either transgenic animals or “knock out” animals that, in turn,are useful in the development and screening of therapeutically usefulreagents. In accordance with established techniques, cDNA encoding151P3D4 can be used to clone genomic DNA that encodes 151P3D4. Thecloned genomic sequences can then be used to generate transgenic animalscontaining cells that express DNA that encode 151P3D4. Methods forgenerating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 issued 12 Apr. 1988, and 4,870,009issued 26 Sep. 1989. Typically, particular cells would be targeted for151P3D4 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 151P3D4can be used to examine the effect of increased expression of DNA thatencodes 151P3D4. Such animals can be used as tester animals for reagentsthought to confer protection from, for example, pathological conditionsassociated with its overexpression. In accordance with this aspect ofthe invention, an animal is treated with a reagent and a reducedincidence of a pathological condition, compared to untreated animalsthat bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 151P3D4 can be used to constructa 151P3D4 “knock out” animal that has a defective or altered geneencoding 151P3D4 as a result of homologous recombination between theendogenous gene encoding 151P3D4 and altered genomic DNA encoding151P3D4 introduced into an embryonic cell of the animal. For example,cDNA that encodes 151P3D4 can be used to clone genomic DNA encoding151P3D4 in accordance with established techniques. A portion of thegenomic DNA encoding 151P3D4 can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 151P3D4 polypeptide.

Methods for the Detection of 151P3D4

Another aspect of the present invention relates to methods for detecting151P3D4 polynucleotides and 151P3D4-related proteins, as well as methodsfor identifying a cell that expresses 151P3D4. The expression profile of151P3D4 makes it a diagnostic marker for metastasized disease.Accordingly, the status of 151P3D4 gene products provides informationuseful for predicting a variety of factors including susceptibility toadvanced stage disease, rate of progression, and/or tumoraggressiveness. As discussed in detail herein, the status of 151P3D4gene products in patient samples can be analyzed by a variety protocolsthat are well known in the art including immunohistochemical analysis,the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of151P3D4 polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 151P3D4 polynucleotides include, for example, a 151P3D4gene or fragment thereof, 151P3D4 mRNA, alternative splice variant151P3D4 mRNAs, and recombinant DNA or RNA molecules that contain a151P3D4 polynucleotide. A number of methods for amplifying and/ordetecting the presence of 151P3D4 polynucleotides are well known in theart and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a 151P3D4 mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a151P3D4 polynucleotides as sense and antisense primers to amplify151P3D4 cDNAs therein; and detecting the presence of the amplified151P3D4 cDNA. Optionally, the sequence of the amplified 151P3D4 cDNA canbe determined.

In another embodiment, a method of detecting a 151P3D4 gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 151P3D4 polynucleotides assense and antisense primers; and detecting the presence of the amplified151P3D4 gene. Any number of appropriate sense and antisense probecombinations can be designed from a 151P3D4 nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a151P3D4 protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 151P3D4-related protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, Westernblot analysis, molecular binding assays, ELISA, ELIFA and the like. Forexample, a method of detecting the presence of a 151P3D4-related proteinin a biological sample comprises first contacting the sample with a151P3D4 antibody, a 151P3D4-reactive fragment thereof, or a recombinantprotein containing an antigen binding region of a 151P3D4 antibody; andthen detecting the binding of 151P3D4-related protein in the sample.

Methods for identifying a cell that expresses 151P3D4 are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 151P3D4 gene comprises detecting the presence of151P3D4 mRNA in the cell. Methods for the detection of particular mRNAsin cells are well known and include, for example, hybridization assaysusing complementary DNA probes (such as in situ hybridization usinglabeled 151P3D4 riboprobes, Northern blot and related techniques) andvarious nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 151P3D4, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like). Alternatively, an assay for identifying a cell that expressesa 151P3D4 gene comprises detecting the presence of 151P3D4-relatedprotein in the cell or secreted by the cell. Various methods for thedetection of proteins are well known in the art and are employed for thedetection of 151P3D4-related proteins and cells that express151P3D4-related proteins.

151P3D4 expression analysis is also useful as a tool for identifying andevaluating agents that modulate 151P3D4 gene expression. For example,151P3D4 expression is significantly upregulated in prostate cancer, andis expressed in cancers of the tissues listed in Table I. Identificationof a molecule or biological agent that inhibits 151P3D4 expression orover-expression in cancer cells is of therapeutic value. For example,such an agent can be identified by using a screen that quantifies151P3D4 expression by RT-PCR, nucleic acid hybridization or antibodybinding.

Methods for Monitoring the Status of 151P3D4-Related Genes and theirProducts

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant151P3D4 expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 151P3D4 in abiological sample of interest can be compared, for example, to thestatus of 151P3D4 in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 151P3D4 in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare151P3D4 status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 151P3D4 expressing cells) as well as the level, andbiological activity of expressed gene products (such as 151P3D4 mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 151P3D4 comprises a change in the location of 151P3D4 and/or151P3D4 expressing cells and/or an increase in 151P3D4 mRNA and/orprotein expression.

151P3D4 status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 151P3D4 geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 151P3D4 in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in a151P3D4 gene), Northern analysis and/or PCR analysis of 151P3D4 mRNA (toexamine, for example alterations in the polynucleotide sequences orexpression levels of 151P3D4 mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 151P3D4 proteins and/orassociations of 151P3D4 proteins with polypeptide binding partners).Detectable 151P3D4 polynucleotides include, for example, a 151P3D4 geneor fragment thereof, 151P3D4 mRNA, alternative splice variants, 151P3D4mRNAs, and recombinant DNA or RNA molecules containing a 151P3D4polynucleotide.

The expression profile of 151P3D4 makes it a diagnostic marker for localand/or metastasized disease, and provides information on the growth oroncogenic potential of a biological sample. In particular, the status of151P3D4 provides information useful for predicting susceptibility toparticular disease stages, progression, and/or tumor aggressiveness. Theinvention provides methods and assays for determining 151P3D4 status anddiagnosing cancers that express 151P3D4, such as cancers of the tissueslisted in Table I. For example, because 151P3D4 mRNA is so highlyexpressed in prostate and other cancers relative to normal prostatetissue, assays that evaluate the levels of 151P3D4 mRNA transcripts orproteins in a biological sample can be used to diagnose a diseaseassociated with 151P3D4 dysregulation, and can provide prognosticinformation useful in defining appropriate therapeutic options.

The expression status of 151P3D4 provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 151P3D4 inbiological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 151P3D4 in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 151P3D4 in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 151P3D4 expressing cells (e.g. those that express151P3D4 mRNAs or proteins). This examination can provide evidence ofdysregulated cellular growth, for example, when 151P3D4-expressing cellsare found in a biological sample that does not normally contain suchcells (such as a lymph node), because such alterations in the status of151P3D4 in a biological sample are often associated with dysregulatedcellular growth. Specifically, one indicator of dysregulated cellulargrowth is the metastases of cancer cells from an organ of origin (suchas the prostate) to a different area of the body (such as a lymph node).In this context, evidence of dysregulated cellular growth is importantfor example because occult lymph node metastases can be detected in asubstantial proportion of patients with prostate cancer, and suchmetastases are associated with known predictors of disease progression(see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al.,Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995August 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 151P3D4gene products by determining the status of 151P3D4 gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 151P3D4gene products in a corresponding normal sample. The presence of aberrant151P3D4 gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 151P3D4 mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 151P3D4 mRNA can, for example, beevaluated in tissues including but not limited to those listed in TableI. The presence of significant 151P3D4 expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 151P3D4mRNA or express it at lower levels.

In a related embodiment, 151P3D4 status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 151P3D4 protein expressed by cells ina test tissue sample and comparing the level so determined to the levelof 151P3D4 expressed in a corresponding normal sample. In oneembodiment, the presence of 151P3D4 protein is evaluated, for example,using immunohistochemical methods. 151P3D4 antibodies or bindingpartners capable of detecting 151P3D4 protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 151P3D4nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 151P3D4 may be indicative of the presence orpromotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 151P3D4 indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 151P3D4 geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. Nos. 5,382,510 issued 7 Sep. 1999, and5,952,170 issued 17 Jan. 1995).

Additionally, one can examine the methylation status of a 151P3D4 genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequently occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prey.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymes thatcannot cleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etal. eds., 1995.

Gene amplification is an additional method for assessing the status of151P3D4. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 151P3D4 expression. The presence of RT-PCRamplifiable 151P3D4 mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting151P3D4 mRNA or 151P3D4 protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 151P3D4 mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 151P3D4 in prostate or other tissue isexamined, with the presence of 151P3D4 in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 151P3D4 nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 151P3D4 gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 151P3D4 mRNA or 151P3D4 proteinexpressed by tumor cells, comparing the level so determined to the levelof 151P3D4 mRNA or 151P3D4 protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 151P3D4 mRNA or 151P3D4 protein expressionin the tumor sample relative to the normal sample indicates the degreeof aggressiveness. In a specific embodiment, aggressiveness of a tumoris evaluated by determining the extent to which 151P3D4 is expressed inthe tumor cells, with higher expression levels indicating moreaggressive tumors. Another embodiment is the evaluation of the integrityof 151P3D4 nucleotide and amino acid sequences in a biological sample,in order to identify perturbations in the structure of these moleculessuch as insertions, deletions, substitutions and the like. The presenceof one or more perturbations indicates more aggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 151P3D4 mRNA or151P3D4 protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 151P3D4 mRNA or 151P3D4 proteinexpressed in an equivalent tissue sample taken from the same individualat a different time, wherein the degree of 151P3D4 mRNA or 151P3D4protein expression in the tumor sample over time provides information onthe progression of the cancer. In a specific embodiment, the progressionof a cancer is evaluated by determining 151P3D4 expression in the tumorcells over time, where increased expression over time indicates aprogression of the cancer. Also, one can evaluate the integrity 151P3D4nucleotide and amino acid sequences in a biological sample in order toidentify perturbations in the structure of these molecules such asinsertions, deletions, substitutions and the like, where the presence ofone or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 151P3D4 gene and151P3D4 gene products (or perturbations in 151P3D4 gene and 151P3D4 geneproducts) and a factor that is associated with malignancy, as a meansfor diagnosing and prognosticating the status of a tissue sample. A widevariety of factors associated with malignancy can be utilized, such asthe expression of genes associated with malignancy (e.g. PSA, PSCA andPSM expression for prostate cancer etc.) as well as gross cytologicalobservations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol.6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al.,1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 151P3D4 gene and 151P3D4 gene products (or perturbationsin 151P3D4 gene and 151P3D4 gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 151P3D4 gene and 151P3D4 gene products (or perturbationsin 151P3D4 gene and 151P3D4 gene products) and another factor associatedwith malignancy entails detecting the overexpression of 151P3D4 mRNA orprotein in a tissue sample, detecting the overexpression of PSA mRNA orprotein in a tissue sample (or PSCA or PSM expression), and observing acoincidence of 151P3D4 mRNA or protein and PSA mRNA or proteinoverexpression (or PSCA or PSM expression). In a specific embodiment,the expression of 151P3D4 and PSA mRNA in prostate tissue is examined,where the coincidence of 151P3D4 and PSA mRNA overexpression in thesample indicates the existence of prostate cancer, prostate cancersusceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 151P3D4 mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 151P3D4 mRNAinclude in situ hybridization using labeled 151P3D4 riboprobes, Northernblot and related techniques using 151P3D4 polynucleotide probes, RT-PCRanalysis using primers specific for 151P3D4, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like. In a specific embodiment, semi-quantitative RT-PCR is usedto detect and quantify 151P3D4 mRNA expression. Any number of primerscapable of amplifying 151P3D4 can be used for this purpose, includingbut not limited to the various primer sets specifically describedherein. In a specific embodiment, polyclonal or monoclonal antibodiesspecifically reactive with the wild-type 151P3D4 protein can be used inan immunohistochemical assay of biopsied tissue.

Identification of Molecules that Interact with 151P3D4

The 151P3D4 protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 151P3D4, as well as pathways activated by 151P3D4 viaany one of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. Nos. 5,955,280 issued 21 Sep. 1999, 5,925,523 issued 20 Jul.1999, 5,846,722 issued 8 Dec. 1998 and 6,004,746 issued 21 Dec. 1999.Algorithms are also available in the art for genome-based predictions ofprotein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999,83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 151P3D4 protein sequences. In such methods, peptidesthat bind to 151P3D4 are identified by screening libraries that encode arandom or controlled collection of amino acids. Peptides encoded by thelibraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the151P3D4 protein(s).

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 151P3D4 proteinsequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued 3Mar. 1998 and 5,733,731 issued 31 Mar. 1998.

Alternatively, cell lines that express 151P3D4 are used to identifyprotein-protein interactions mediated by 151P3D4. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 151P3D4protein can be immunoprecipitated from 151P3D4-expressing cell linesusing anti-151P3D4 antibodies. Alternatively, antibodies against His-tagcan be used in a cell line engineered to express fusions of 151P3D4 anda His-tag (vectors mentioned above). The immunoprecipitated complex canbe examined for protein association by procedures such as Westernblotting, ³⁵S-methionine labeling of proteins, protein microsequencing,silver staining and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with 151P3D4 can be identifiedthrough related embodiments of such screening assays. For example, smallmolecules can be identified that interfere with protein function,including molecules that interfere with 151P3D4's ability to mediatephosphorylation and de-phosphorylation, interaction with DNA or RNAmolecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 151P3D4-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 151P3D4 (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 151P3D4 function can beidentified based on their ability to bind 151P3D4 and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of151P3D4 and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators which activate or inhibit 151P3D4.

An embodiment of this invention comprises a method of screening for amolecule that interacts with a 151P3D4 amino acid sequence shown in FIG.2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 151P3D4 amino acid sequence, allowing the population ofmolecules and the 151P3D4 amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 151P3D4 amino acid sequence, and thenseparating molecules that do not interact with the 151P3D4 amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 151P3D4 amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 151P3D4. In apreferred embodiment, the 151P3D4 amino acid sequence is contacted witha library of peptides.

Therapeutic Methods and Compositions

The identification of 151P3D4 as a protein that is normally expressed ina restricted set of tissues, but which is also expressed in prostate andother cancers, opens a number of therapeutic approaches to the treatmentof such cancers. As contemplated herein, 151P3D4 functions as atranscription factor involved in activating tumor-promoting genes orrepressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a151P3D4 protein are useful for patients suffering from a cancer thatexpresses 151P3D4. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of a 151P3D4 protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of a 151P3D4 gene or translation of 151P3D4mRNA.

Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 151P3D4-relatedprotein or 151P3D4-related nucleic acid. In view of the expression of151P3D4, cancer vaccines prevent and/or treat 151P3D4-expressing cancerswith minimal or no effects on non-target tissues. The use of a tumorantigen in a vaccine that generates humoral and/or cell-mediated immuneresponses as anti-cancer therapy is well known in the art and has beenemployed in prostate cancer using human PSMA and rodent PAP immunogens(Hodge et al., 1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J.Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 151P3D4-relatedprotein, or a 151P3D4-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 151P3D4 immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al.,Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methodsof generating an immune response (e.g. humoral and/or cell-mediated) ina mammal, comprise the steps of: exposing the mammal's immune system toan immunoreactive epitope (e.g. an epitope present in a 151P3D4 proteinshown in FIG. 3 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, a 151P3D4 immunogen contains a biological motif, seee.g., Tables V-XVIII and XXII-LI, or a peptide of a size range from151P3D4 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 151P3D4 protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 151P3D4-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 151P3D4 protein that bind corresponding HLA alleles (seee.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL located onthe World Wide Web at(.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html)); and, BIMAS,(URL bimas.dcrt.nih.gov/; SYFPEITHI at URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a 151P3D4immunogen contains one or more amino acid sequences identified usingtechniques well known in the art, such as the sequences shown in TablesV-XVIII and XXII-LI or a peptide of 8, 9, 10 or 11 amino acids specifiedby an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), orTable IV (E)) and/or a peptide of at least 9 amino acids that comprisesan HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)).As is appreciated in the art, the HLA Class I binding groove isessentially closed ended so that peptides of only a particular sizerange can fit into the groove and be bound, generally HLA Class Iepitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLAClass II binding groove is essentially open ended; therefore a peptideof about 9 or more amino acids can be bound by an HLA Class II molecule.Due to the binding groove differences between HLA Class I and II, HLAClass I motifs are length specific, i.e., position two of a Class Imotif is the second amino acid in an amino to carboxyl direction of thepeptide. The amino acid positions in a Class II motif are relative onlyto each other, not the overall peptide, i.e., additional amino acids canbe attached to the amino and/or carboxyl termini of a motif-bearingsequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. a 151P3D4 protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 151P3D4 in a host, by contacting the host with asufficient amount of at least one 151P3D4 B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 151P3D4 B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 151P3D4-related protein or aman-made multiepitopic peptide comprising: administering 151P3D4immunogen (e.g. a 151P3D4 protein or a peptide fragment thereof, a151P3D4 fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 151P3D4 immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes a 151P3D4 immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 151P3D4, in order to generate a response to thetarget antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 151P3D4. Constructscomprising DNA encoding a 151P3D4-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 151P3D4 protein/immunogen.Alternatively, a vaccine comprises a 151P3D4-related protein. Expressionof the 151P3D4-related protein immunogen results in the generation ofprophylactic or therapeutic humoral and cellular immunity against cellsthat bear a 151P3D4 protein. Various prophylactic and therapeuticgenetic immunization techniques known in the art can be used (forreview, see information and references published at Internet addresslocated on the World Wide Web at .genweb.com). Nucleic acid-baseddelivery is described, for instance, in Wolff et. al., Science 247:1465(1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566;5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-baseddelivery technologies include “naked DNA”, facilitated (bupivicaine,polymers, peptide-mediated) delivery, cationic lipid complexes, andparticle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g.,U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 151P3D4-related protein into the patient (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmette Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 151P3D4-relatednucleic acid molecule. In one embodiment, the full-length human 151P3D4cDNA is employed. In another embodiment, 151P3D4 nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCs) such as dendritic cells (DC) to present 151P3D4 antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 151P3D4 peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with151P3D4 peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 151P3D4 protein. Yet another embodiment involves engineeringthe overexpression of a 151P3D4 gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 151P3D4 can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

151P3D4 as a Target for Antibody-Based Therapy

151P3D4 is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 151P3D4 is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of151P3D4-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 151P3D4 areuseful to treat 151P3D4-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

151P3D4 antibodies can be introduced into a patient such that theantibody binds to 151P3D4 and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 151P3D4,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 151P3D4 sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 151P3D4), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-151P3D4 antibody) that binds to a marker (e.g. 151P3D4)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 151P3D4, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a151P3D4 epitope, and, exposing the cell to the antibody-agent conjugate.Another illustrative embodiment is a method of treating an individualsuspected of suffering from metastasized cancer, comprising a step ofadministering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-151P3D4 antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 151P3D4 antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG₄kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 151P3D4 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Although 151P3D4 antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 151P3D4expression, preferably using immunohistochemical assessments of tumortissue, quantitative 151P3D4 imaging, or other techniques that reliablyindicate the presence and degree of 151P3D4 expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-151P3D4 monoclonal antibodies that treat prostate and other cancersinclude those that initiate a potent immune response against the tumoror those that are directly cytotoxic. In this regard, anti-151P3D4monoclonal antibodies (mAbs) can elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-151P3D4 mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 151P3D4. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-151P3D4 mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 151P3D4antigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-151P3D4 mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-151P3D4 mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-151P3D4 mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-151P3D4 antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of the anti-151P3D4antibody preparation, via an acceptable route of administration such asintravenous injection (IV), typically at a dose in the range of about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of10-1000 mg mAb per week are effective and well tolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-151P3D4 mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose is administeredas a 30 minute or longer infusion, provided the initial dose was welltolerated. As appreciated by those of skill in the art, various factorscan influence the ideal dose regimen in a particular case. Such factorsinclude, for example, the binding affinity and half life of the Ab ormAbs used, the degree of 151P3D4 expression in the patient, the extentof circulating shed 151P3D4 antigen, the desired steady-state antibodyconcentration level, frequency of treatment, and the influence ofchemotherapeutic or other agents used in combination with the treatmentmethod of the invention, as well as the health status of a particularpatient.

Optionally, patients should be evaluated for the levels of 151P3D4 in agiven sample (e.g. the levels of circulating 151P3D4 antigen and/or151P3D4 expressing cells) in order to assist in the determination of themost effective dosing regimen, etc. Such evaluations are also used formonitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-151P3D4 antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 151P3D4-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-151P3D4 antibodiesthat mimic an epitope on a 151P3D4-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

151P3D4 as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis, J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 151P3D4 antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TAA). For HLA Class II a similar rationale isemployed; again 3-4 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif- and/or motif-bearing epitopes derived 151P3D4, the PADRE®universal helper T cell epitope or multiple HTL epitopes from 151P3D4(see e.g., Tables V-XVIII and XXII to LI), and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE®, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al.,Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:44), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 45), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 46). Otherexamples include peptides bearing a DR 1-4-7 supermotif, or either ofthe DR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed to most preferably bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: aKXVAAWTLKAAa (SEQ ID NO: 47), where “X” is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to specifically primean immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP₃CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 151P3D4. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 151P3D4.

Adoptive Immunotherapy

Antigenic 151P3D4-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses151P3D4. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 151P3D4. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 151P3D4-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 151P3D4, a vaccine comprising 151P3D4-specific CTL may bemore efficacious in killing tumor cells in patient with advanced diseasethan alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to effectivelystimulate a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μg,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-151P3D4 antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg mAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-151P3D4 mAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of 151P3D4 expression inthe patient, the extent of circulating shed 151P3D4 antigen, the desiredsteady-state concentration level, frequency of treatment, and theinfluence of chemotherapeutic or other agents used in combination withthe treatment method of the invention, as well as the health status of aparticular patient. Non-limiting preferred human unit doses are, forexample, 500 μg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg,800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses;0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body areaweekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01%-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

Diagnostic and Prognostic Embodiments of 151P3D4.

As disclosed herein, 151P3D4 polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in the Example entitled “Expression analysis of 151P3D4 innormal tissues, and patient specimens”).

151P3D4 can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640 (1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto etal., Cancer Detect Prey 2000; 24(1):1-12). Therefore, this disclosure of151P3D4 polynucleotides and polypeptides (as well as 151P3D4polynucleotide probes and anti-151P3D4 antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 151P3D4polynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays whichemploy, e.g., PSA polynucleotides, polypeptides, reactive T cells andantibodies. For example, just as PSA polynucleotides are used as probes(for example in Northern analysis, see, e.g., Sharief et al., Biochem.Mol. Biol. Int. 33(3):567-74 (1994)) and primers (for example in PCRanalysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000))to observe the presence and/or the level of PSA mRNAs in methods ofmonitoring PSA overexpression or the metastasis of prostate cancers, the151P3D4 polynucleotides described herein can be utilized in the same wayto detect 151P3D4 overexpression or the metastasis of prostate and othercancers expressing this gene. Alternatively, just as PSA polypeptidesare used to generate antibodies specific for PSA which can then be usedto observe the presence and/or the level of PSA proteins in methods tomonitor PSA protein overexpression (see, e.g., Stephan et al., Urology55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g.,Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 151P3D4polypeptides described herein can be utilized to generate antibodies foruse in detecting 151P3D4 overexpression or the metastasis of prostatecells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 151P3D4polynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 151P3D4-expressing cells (lymph node) is found tocontain 151P3D4-expressing cells such as the 151P3D4 expression seen inLAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 151P3D4 polynucleotides and/or polypeptides can be used toprovide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 151P3D4 or express 151P3D4 at adifferent level are found to express 151P3D4 or have an increasedexpression of 151P3D4 (see, e.g., the 151P3D4 expression in the cancerslisted in Table I and in patient samples etc. shown in the accompanyingFigures). In such assays, artisans may further wish to generatesupplementary evidence of metastasis by testing the biological samplefor the presence of a second tissue restricted marker (in addition to151P3D4) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res.Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,151P3D4 polynucleotide fragments and polynucleotide variants are used inan analogous manner. In particular, typical PSA polynucleotides used inmethods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in the Example entitled “Expression analysisof 151P3D4 in normal tissues, and patient specimens,” where a 151P3D4polynucleotide fragment is used as a probe to show the expression of151P3D4 RNAs in cancer cells. In addition, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see, e.g., Sawai et al., FetalDiagn. Ther. 1996 November-December 11(6):407-13 and Current ProtocolsIn Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al.eds., 1995)). Polynucleotide fragments and variants are useful in thiscontext where they are capable of binding to a target polynucleotidesequence (e.g., a 151P3D4 polynucleotide shown in FIG. 2 or variantthereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 151P3D4 polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 151P3D4biological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 151P3D4 polypeptide shown in FIG. 3).

As shown herein, the 151P3D4 polynucleotides and polypeptides (as wellas the 151P3D4 polynucleotide probes and anti-151P3D4 antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of151P3D4 gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 151P3D4polynucleotides and polypeptides (as well as the 151P3D4 polynucleotideprobes and anti-151P3D4 antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 151P3D4polynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 151P3D4 gene maps(see the Example entitled “Chromosomal Mapping of 151P3D4” below).Moreover, in addition to their use in diagnostic assays, the151P3D4-related proteins and polynucleotides disclosed herein have otherutilities such as their use in the forensic analysis of tissues ofunknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28;80(1-2): 63-9).

Additionally, 151P3D4-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 151P3D4. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 151P3D4 antigen. Antibodies or othermolecules that react with 151P3D4 can be used to modulate the functionof this molecule, and thereby provide a therapeutic benefit.

Inhibition of 151P3D4 Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 151P3D4 to its binding partner or its association withother protein(s) as well as methods for inhibiting 151P3D4 function.

Inhibition of 151P3D4 with Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 151P3D4 are introduced into 151P3D4expressing cells via gene transfer technologies. Accordingly, theencoded single chain anti-151P3D4 antibody is expressed intracellularly,binds to 151P3D4 protein, and thereby inhibits its function. Methods forengineering such intracellular single chain antibodies are well known.Such intracellular antibodies, also known as “intrabodies”, arespecifically targeted to a particular compartment within the cell,providing control over where the inhibitory activity of the treatment isfocused. This technology has been successfully applied in the art (forreview, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodieshave been shown to virtually eliminate the expression of otherwiseabundant cell surface receptors (see, e.g., Richardson et al., 1995,Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol.Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to precisely target the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 151P3D4 in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 151P3D4 intrabodies in orderto achieve the desired targeting. Such 151P3D4 intrabodies are designedto bind specifically to a particular 151P3D4 domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a 151P3D4protein are used to prevent 151P3D4 from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 151P3D4 from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).

Inhibition of 151P3D4 with Recombinant Proteins

In another approach, recombinant molecules bind to 151P3D4 and therebyinhibit 151P3D4 function. For example, these recombinant moleculesprevent or inhibit 151P3D4 from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 151P3D4specific antibody molecule. In a particular embodiment, the 151P3D4binding domain of a 151P3D4 binding partner is engineered into a dimericfusion protein, whereby the fusion protein comprises two 151P3D4 ligandbinding domains linked to the Fc portion of a human IgG, such as humanIgG1. Such IgG portion can contain, for example, the C_(H)2 and C_(H)3domains and the hinge region, but not the C_(H)1 domain. Such dimericfusion proteins are administered in soluble form to patients sufferingfrom a cancer associated with the expression of 151P3D4, whereby thedimeric fusion protein specifically binds to 151P3D4 and blocks 151P3D4interaction with a binding partner. Such dimeric fusion proteins arefurther combined into multimeric proteins using known antibody linkingtechnologies.

Inhibition of 151P3D4 Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 151P3D4 gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 151P3D4 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 151P3D4gene comprises contacting the 151P3D4 gene with a 151P3D4 antisensepolynucleotide. In another approach, a method of inhibiting 151P3D4 mRNAtranslation comprises contacting a 151P3D4 mRNA with an antisensepolynucleotide. In another approach, a 151P3D4 specific ribozyme is usedto cleave a 151P3D4 message, thereby inhibiting translation. Suchantisense and ribozyme based methods can also be directed to theregulatory regions of the 151P3D4 gene, such as 151P3D4 promoter and/orenhancer elements. Similarly, proteins capable of inhibiting a 151P3D4gene transcription factor are used to inhibit 151P3D4 mRNAtranscription. The various polynucleotides and compositions useful inthe aforementioned methods have been described above. The use ofantisense and ribozyme molecules to inhibit transcription andtranslation is well known in the art.

Other factors that inhibit the transcription of 151P3D4 by interferingwith 151P3D4 transcriptional activation are also useful to treat cancersexpressing 151P3D4. Similarly, factors that interfere with 151P3D4processing are useful to treat cancers that express 151P3D4. Cancertreatment methods utilizing such factors are also within the scope ofthe invention.

General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing 151P3D4(i.e., antisense, ribozyme, polynucleotides encoding intrabodies andother 151P3D4 inhibitory molecules). A number of gene therapy approachesare known in the art. Recombinant vectors encoding 151P3D4 antisensepolynucleotides, ribozymes, factors capable of interfering with 151P3D4transcription, and so forth, can be delivered to target tumor cellsusing such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 151P3D4 to a bindingpartner, etc.

In vivo, the effect of a 151P3D4 therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a 151P3D4-related protein or a 151P3D4 geneor message, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, and canalso indicate directions for either in vivo or in vitro use, such asthose described above. Directions and or other information can also beincluded on an insert which is included with the kit.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of a cDNA Fragment of the 151P3D4 Gene

To isolate genes that are over-expressed in bladder cancer we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom the LNCaP prostate cancer cell line.

The 151P3D4 SSH cDNA sequence was derived from a subtraction consistingof two different populations of LNCaP cells. The 151P3D4 SSH cDNAsequence of 417 by is listed in FIG. 1.

The full-length 151P3D4 v.1 clone 1-placenta was cloned from normalplacenta cDNA, revealing an ORF of 354 amino acids (FIG. 2 and FIG. 3).Other variants of 151P3D4 were also identified and these are listed inFIGS. 2 and 3.

Materials and Methods

Human Tissues:

The patient cancer and normal tissues were purchased from differentsources such as the NDRI (Philadelphia, Pa.). mRNA for some normaltissues were purchased from Clontech, Palo Alto, Calif.

RNA Isolation:

Tissues were homogenized in Trizol reagent (Life Technologies, GibcoBRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purifiedfrom total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Totaland mRNA were quantified by spectrophotometric analysis (O.D. 260/280nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): (SEQ ID NO: 48) 5′TTTTGATCAAGCTT₃₀3′Adaptor 1: (SEQ ID NO: 49)5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO: 50)3′GGCCCGTCCTAG5′ Adaptor 2: (SEQ ID NO: 51)5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 52)3′CGGCTCCTAG5′ PCR primer 1: (SEQ ID NO: 53) 5′CTAATACGACTCACTATAGGGC3′Nested primer (NP)1: (SEQ ID NO: 54) 5′TCGAGCGGCCGCCCGGGCAGGA3′ Nestedprimer (NP)2: (SEQ ID NO: 55) 5′AGCGTGGTCGCGGCCGAGGA3′

Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from two different clones ofLNCaP cells.

The gene 151P3D4 was derived from one population of LNCaP cells minusanother population of LNCaP cells cDNA subtraction. The 151P3D4 SSH DNAsequence (FIG. 1) was identified.

The cDNA derived from one population of LNCaP cells was used as thesource of the “driver” cDNA, while the cDNA from another population ofLNCaP cells was used as the source of the “tester” cDNA. Double strandedcDNAs corresponding to tester and driver cDNAs were synthesized from 2μg of poly(A)⁺ RNA isolated from the relevant tissue, as describedabove, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng ofoligonucleotide DPNCDN as primer. First- and second-strand synthesiswere carried out as described in the Kit's user manual protocol(CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resultingcDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA wasextracted with phenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA was generated by combining in a 1:1 ratio Dpn II digestedcDNA from the relevant source (see above). Tester cDNA was generated bydiluting 1 μl of Dpn II digested cDNA from the relevant source (seeabove) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) wasthen ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separateligation reactions, in a total volume of 10 μl at 16° C. overnight,using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 the sampleswere overlaid with mineral oil, denatured in an MJ Research thermalcycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for8 hrs at 68° C. The two hybridizations were then mixed together with anadditional 1 μl of fresh denatured driver cDNA and were allowed tohybridize overnight at 68° C. The second hybridization was then dilutedin 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70°C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′ atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO: 56) and 5′ agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 57) to amplifyβ-actin. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 b.p.β-actin bands from multiple tissues were compared by visual inspection.Dilution factors for the first strand cDNAs were calculated to result inequal β-actin band intensities in all tissues after 22 cycles of PCR.Three rounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 151P3D4 gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities. The primers used for RT-PCR were designed using the 151P3D4SSH sequence and are listed below:

151P3D4.1 5′- CCCACCAAACTGACCTATGATGAA -3′ (SEQ ID NO: 58) 151P3D4.2 5′-TGTATGCTCTGAAGCAGTAGACACC -3′ (SEQ ID NO: 59)

A typical RT-PCR expression study is shown in FIG. 14. First strand cDNAwas prepared from vital pool 1 (liver, lung and kidney), vital pool 2(pancreas, colon and stomach), bladder cancer pool, kidney cancer pool,colon cancer pool, lung cancer pool, ovary cancer pool, breast cancerpool, and cancer metastasis pool. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto 151P3D4, was performed at 26 and 30 cycles of amplification. Resultsshow strong expression of 151P3D4 in ovary cancer pool. Expression of151P3D4 was also detected in bladder cancer pool, kidney cancer pool,colon cancer pool, lung cancer pool, breast cancer pool, cancermetastasis pool, vital pool 2, but not in vital pool 1.

Example 2 Full Length Cloning of 151P3D4

To isolate genes that are expressed in prostate cancer, we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom two different populations of LNCaP cells.

The 151P3D4 SSH cDNA sequence was derived from a subtraction consistingof one population of LNCaP cells minus another population of LNCaPcells. The 151P3D4 SSH cDNA sequence of 417 bp is listed in FIG. 1.

The full-length 151P3D4 v.1 (151P3D4 clone 1-placenta) was cloned fromnormal placenta cDNA, revealing an ORF of 354 amino acids (FIG. 2 andFIG. 3). 151P3D4 v.1 showed 99% identity over 1492 nucleotides with thehuman mRNA for cartilage link protein (gi463246) (FIG. 4A). 151P3D4 v.1protein showed 100% identity over 354 amino acids with the humancartilage link protein (FIG. 4B). Also, 151P3D4 v.1 was 96% identicalover 355 amino acids with the mouse link protein (gi4218976) (FIG. 4C).

Other variants of 151P3D4 were also identified and these are listed inFIGS. 2 and 3. 151P3D4 v.2 codes for a novel protein that containssequences not present in 151P3D4 v.1. These are from amino acids 1 to400. Amino acids 401 to 721 of 151P3D4 v.2 align with 151P3D4 v.1 atpositions 34 to 354 (FIG. 4D). A small portion of 151P3D4 v.2demonstrates homology to the hypothetical protein XP_(—)094318 (FIG.4E). The two proteins show 99% identity over 168 amino acids. The othervariants 151P3D4 v.3 through v.11 each differ from 151P3D4 v.1 by onenucleotide (FIG. 10).

Example 3 Chromosomal Mapping of 151P3D4

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville, Ala.), human-rodent somatic cell hybrid panelssuch as is available from the Coriell Institute (Camden, N.J.), andgenomic viewers utilizing BLAST homologies to sequenced and mappedgenomic clones (NCBI, Bethesda, Md.).

151P3D4 maps to chromosome 5q13-q14.1 using 151P3D4 sequence and theNCBI BLAST tool: located on the World Wide Web at(.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs).

Example 4 Expression Analysis of 151P3D4 in Normal Tissues and PatientSpecimens

Expression analysis by RT-PCR demonstrated that 151P3D4 is stronglyexpressed in cancer patient specimens (FIG. 14). First strand cDNA wasprepared from vital pool 1 (liver, lung and kidney), vital pool 2(pancreas, colon and stomach), bladder cancer pool, kidney cancer pool,colon cancer pool, lung cancer pool, ovary cancer pool, breast cancerpool, and cancer metastasis pool. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto 151P3D4, was performed at 26 and 30 cycles of amplification. Resultsshow strong expression of 151P3D4 in ovary cancer pool. Expression of151P3D4 was also detected in bladder cancer pool, kidney cancer pool,colon cancer pool, lung cancer pool, breast cancer pool, cancermetastasis pool, vital pool 2, but not in vital pool 1.

Extensive northern blot analysis of 151P3D4 in multiple human normaltissues is shown in FIG. 15. Two multiple tissue northern blots(Clontech) both with 2 ug of mRNA/lane were probed with the 151P3D4 SSHsequence. Size standards in kilobases (kb) are indicated on the side.Results show expression of 151P3D4 in small intestine and placenta.Lower level expression was also detected in heart and colon, but not inthe other normal tissues tested.

Expression of 151P3D4 in patient bladder cancer specimens is shown inFIG. 16. RNA was extracted from normal bladder (NB), bladder cancer celllines (CL; UM-UC-3, J82, SCaBER), bladder cancer patient tumors (T) andnormal adjacent tissue (NAT). Northern blots with 10 ug of total RNAwere probed with the 151P3D4 SSH sequence. Size standards in kilobasesare indicated on the side. Results show expression of 151P3D4 in patientbladder cancer tissues, and in UM-UC-3 bladder cancer cell lines, butnot in normal bladder nor in the other bladder cancer cell lines tested.

FIG. 17 shows that 151P3D4 was expressed in kidney cancer patientspecimens. RNA was extracted from kidney cancer cell lines (CL: 769-P,A498, SW839), normal kidney (NK), kidney cancer patient tumors (T) andtheir normal adjacent tissues (NAT). Northern blots with 10 ug of totalRNA were probed with the 151P3D4 SSH sequence. Size standards inkilobases are on the side. Results show expression of 151P3D4 in patientkidney tumor tissues, but not in normal kidney, nor in the cell linestested.

Expression of 151P3D4 was also detected in ovary cancer patient specimen(FIG. 18). RNA was extracted from ovary and cervical cancer cell lines(CL), normal ovary (N), and ovary cancer patient tumor (T). Northernblots with 10 ug of total RNA were probed with the 151P3D4 SSH sequence.Size standards in kilobases are on the side. Results show strongexpression of 151P3D4 in patient ovary cancer tissues, but not in normalovary nor in the ovary and cervical cancer cell lines.

FIG. 19 shows that 151P3D4 was also expressed in stomach cancers and inuterus cancers. Expression of 151P3D4 was assayed in a panel of humanstomach and uterus cancers (T) and their respective matched normaltissues (N) on RNA dot blots. 151P3D4 expression was seen in bothstomach and uterus cancers.

The restricted expression of 151P3D4 in normal tissues and theexpression detected in human cancers suggest that 151P3D4 is a potentialtherapeutic target and a diagnostic marker for human cancers.

Example 5 Transcript Variants of 151P3D4

Transcript variants are variants of matured mRNA from the same gene byalternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or mayencode proteins with different functions, and may be expressed in thesame tissue at the same time, or at different tissue, or at differenttimes, proteins encoded by transcript variants can have similar ordifferent cellular or extracellular localizations, i.e., be secreted.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedin a full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene (see, e.g., thewebsite located on the World Wide Web at(.doubletwist.com/products/c11_agentsOverview.jhtml)). Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. 2000 April; 10(4):516-22); Grail Internet website(compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan Internet website(genes.mit.edu/GENSCAN.html). For a general discussion of splice variantidentification protocols see., e.g., Southan, C., A genomic perspectiveon human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S.J., et al., Identification of human chromosome 22 transcribed sequenceswith ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7;97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. 1997 Aug. 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 151P3D4 has aparticular expression profile. Alternative transcripts and splicevariants of 151P3D4 that are structurally and/or functionally similar to151P3D4 share this expression pattern, thus serving as tumor associatedmarkers/antigens.

The exon composition of the original transcript, designated as 151P3D4v.1, is shown in Table LII (A). Using the full-length gene and ESTsequences, one alternative transcript was identified, designated as151P3D4 v.2. Compared with 151P3D4 v.1, transcript variant 151P3D4 v.2has 10 exons, as shown in Table LII (B) and FIG. 12. Exons 8 and 9 arethe same as exons 3 and 4 of 151P3D4 v.1, and exon 10 is the codingportion of exon 5 of 151P3D4 v.1. Each different combination of exons inspatial order, e.g. exons 2 and 3, is a potential splice variant. FIG.12 shows the schematic alignment of exons of the two transcriptvariants.

Table LIII shows nucleotide sequence of the transcript variant, 151P3D4v.2 (see also FIG. 2B). Table LIV shows the alignment of the transcriptvariant 151P3D4 v.2 with nucleic acid sequence of 151P3D4 v.1. FIG. 3Bprovides the amino acid translation of the transcript variant 151P3D4v.2 for the identified reading frame orientation. Table LV displaysalignments of the amino acid sequence encoded by the transcript variant151P3D4 v.2 with that of 151P3D4 v.1.

Example 6 Single Nucleotide Polymorphisms of 151P3D4

Single Nucleotide Polymorphism (SNP) is a single base pair variation innucleotide sequences. At a specific point of the genome, there are fourpossible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refersto the base pair make-up of one or more spots in the genome of anindividual, while haplotype refers to base pair make-up of more than onevaried spots on the same DNA molecule (chromosome in higher organism).SNPs that occur on a cDNA are called cSNPs. These cSNPs may change aminoacids of the protein encoded by the gene and thus change the functionsof the protein. Some SNPs cause inherited diseases and some otherscontribute to quantitative variations in phenotype and reactions toenvironmental factors including diet and drugs among individuals.Therefore, SNPs and/or combinations of alleles (called haplotypes) havemany applications including diagnosis of inherited diseases,determination of drug reactions and dosage, identification of genesresponsible for diseases and discovery of genetic relationship betweenindividuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis todissect human traits,” Curr. Opin. Neurobiol. 2001 October;11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility toadverse drug reactions,” Trends Pharmacol. Sci. 2001 June;22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The useof single nucleotide polymorphisms in the isolation of common diseasegenes,” Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C.Stephens and A. Windemuth, “The predictive power of haplotypes inclinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab. 2001October-November; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 February;47(2):164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.2000 December; 5(4):329-340).

Using the methods described above, nine SNPs were identified in theoriginal transcript, 151P3D4 v.1, at positions 154 (A/G), 218 (C/G), 219(G/C), 999 (C/G), 1326 (C/T), 1399 (T/C), 1400 (C/T), 1653 (T/C) and1726 (A/G). The transcripts or proteins with alternative alleles weredesignated as variants 151P3D4 v.3, v.4, v.5, v.6, v.7, v.8, v.9, v.10and v.11. FIGS. 10 and 12 show the schematic alignment of the nucleotidevariants. FIG. 11 shows the schematic alignment of protein variants,corresponding to nucleotide variants. Nucleotide variants that code forthe same amino acid sequence as variant 1 are not shown in FIG. 11.These alleles of the SNPs, though shown separately here, can occur indifferent combinations (haplotypes) and in any one of the transcriptvariants that contains the sequence context of the SNPs, e.g., 151P3D4v.7.

Example 7 Production of Recombinant 151P3D4 in Prokaryotic Systems

To express recombinant 151P3D4 and 151P3D4 variants in prokaryoticcells, the full or partial length 151P3D4 and 151P3D4 variant cDNAsequences are cloned into any one of a variety of expression vectorsknown in the art. One or more of the following regions of 151P3D4variants are expressed: the full length sequence presented in FIGS. 2and 3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from151P3D4, variants, or analogs thereof.

A. In Vitro Transcription and Translation Constructs:

pCRII: To generate 151P3D4 sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of the 151P3D4 cDNA. ThepCRII vector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 151P3D4 RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 151P3D4 at the RNA level. Transcribed 151P3D4 RNArepresenting the cDNA amino acid coding region of the 151P3D4 gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize151P3D4 protein.

B. Bacterial Constructs:

pGEX Constructs: To generate recombinant 151P3D4 proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of the 151P3D4 cDNA protein coding sequence are cloned into thepGEX family of GST-fusion vectors (Amersham Pharmacia Biotech,Piscataway, N.J.). These constructs allow controlled expression ofrecombinant 151P3D4 protein sequences with GST fused at theamino-terminus and a six histidine epitope (6× His) at thecarboxyl-terminus. The GST and 6× His tags permit purification of therecombinant fusion protein from induced bacteria with the appropriateaffinity matrix and allow recognition of the fusion protein withanti-GST and anti-His antibodies. The 6× His tag is generated by adding6 histidine codons to the cloning primer at the 3′ end, e.g., of theopen reading frame (ORF). A proteolytic cleavage site, such as thePreScission™ recognition site in pGEX-6P-1, may be employed such that itpermits cleavage of the GST tag from 151P3D4-related protein. Theampicillin resistance gene and pBR322 origin permits selection andmaintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 151P3D4 proteinsthat are fused to maltose-binding protein (MBP), all or parts of the151P3D4 cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 151P3D4 protein sequences with MBP fused at theamino-terminus and a 6× His epitope tag at the carboxyl-terminus. TheMBP and 6× His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6× His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 151P3D4. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

pET Constructs: To express 151P3D4 in bacterial cells, all or parts ofthe 151P3D4 cDNA protein coding sequence are cloned into the pET familyof vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 151P3D4 protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6× His and S-Tag™ that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of the 151P3D4 protein are expressed as amino-terminal fusionsto NusA.

C. Yeast Constructs:

pESC Constructs: To express 151P3D4 in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of the 151P3D4 cDNA protein coding sequence are cloned intothe pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either Flag™ or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 151P3D4. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 151P3D4 in the yeast species Saccharomycespombe, all or parts of the 151P3D4 cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 151P3D4 protein sequence that is fused ateither the amino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 151P3D4 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant 151P3D4 in eukaryotic cells, the full or partiallength 151P3D4 cDNA sequences can be cloned into any one of a variety ofexpression vectors known in the art. One or more of the followingregions of 151P3D4 are expressed in these constructs, amino acids 1 to354 of 151P3D4 v.1, amino acids 1 to 721 of 151P3D4 v.2, or any 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50 or more contiguous amino acids from 151P3D4,variants, or analogs thereof. In certain embodiments a region of aspecific variant of 151P3D4 is expressed that encodes an amino acid at aspecific position which differs from the amino acid of any other variantfound at that position. In other embodiments, a region of a variant of151P3D4 is expressed that lies partly or entirely within a sequence thatis unique to that variant.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-151P3D4 polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 151P3D4 in mammalian cells, a151P3D4 ORF, or portions thereof, of 151P3D4 are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has Xpress™ and sixhistidine (6× His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.1/MycHis Constructs: To express 151P3D4 in mammalian cells, a151P3D4 ORF, or portions thereof, of 151P3D4 with a consensus Kozaktranslation initiation site was cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant protein has the mycepitope and 6× His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene was used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli. Results of expression from 151P3D4 .pcDNA3.1/MycHisconstruct are shown in FIG. 20.

pcDNA3.1/CT-GFP-TOPO Construct: To express 151P3D4 in mammalian cellsand to allow detection of the recombinant proteins using fluorescence, a151P3D4 ORF, or portions thereof, with a consensus Kozak translationinitiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA).Protein expression is driven from the cytomegalovirus (CMV) promoter.The recombinant proteins have the Green Fluorescent Protein (GFP) fusedto the carboxyl-terminus facilitating non-invasive, in vivo detectionand cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also containsthe bovine growth hormone (BGH) polyadenylation signal and transcriptiontermination sequence to enhance mRNA stability along with the SV40origin for episomal replication and simple vector rescue in cell linesexpressing the large T antigen. The Neomycin resistance gene allows forselection of mammalian cells that express the protein, and theampicillin resistance gene and ColE1 origin permits selection andmaintenance of the plasmid in E. coli. Additional constructs with anamino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning theentire length of a 151P3D4 protein.

PAPtag: A 151P3D4 ORF, or portions thereof, is cloned into pAPtag-5(GenHunter Corp. Nashville, Tenn.). This construct generates an alkalinephosphatase fusion at the carboxyl-terminus of a 151P3D4 protein whilefusing the IgGK signal sequence to the amino-terminus. Constructs arealso generated in which alkaline phosphatase with an amino-terminal IgGKsignal sequence is fused to the amino-terminus of a 151P3D4 protein. Theresulting recombinant 151P3D4 proteins are optimized for secretion intothe media of transfected mammalian cells and can be used to identifyproteins such as ligands or receptors that interact with 151P3D4proteins. Protein expression is driven from the CMV promoter and therecombinant proteins also contain myc and 6× His epitopes fused at thecarboxyl-terminus that facilitates detection and purification. TheZeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the recombinant protein and the ampicillinresistance gene permits selection of the plasmid in E. coli.

ptag5: A 151P3D4 ORF, or portions thereof, is cloned into pTag-5. Thisvector is similar to pAPtag but without the alkaline phosphatase fusion.This construct generates 151P3D4 protein with an amino-terminal IgGκsignal sequence and myc and 6× His epitope tags at the carboxyl-terminusthat facilitate detection and affinity purification. The resultingrecombinant 151P3D4 protein is optimized for secretion into the media oftransfected mammalian cells, and is used as immunogen or ligand toidentify proteins such as ligands or receptors that interact with the151P3D4 proteins. Protein expression is driven from the CMV promoter.The Zeocin resistance gene present in the vector allows for selection ofmammalian cells expressing the protein, and the ampicillin resistancegene permits selection of the plasmid in E. coli.

PsecFc: A 151P3D4 ORF, or portions thereof, is also cloned into psecFc.The psecFc vector was assembled by cloning the human immunoglobulin G1(IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen,California). This construct generates an IgG1 Fc fusion at thecarboxyl-terminus of the 151P3D4 proteins, while fusing the IgGK signalsequence to N-terminus. 151P3D4 fusions utilizing the murine IgG1 Fcregion are also used. The resulting recombinant 151P3D4 proteins areoptimized for secretion into the media of transfected mammalian cells,and can be used as immunogens or to identify proteins such as ligands orreceptors that interact with 151P3D4 protein. Protein expression isdriven from the CMV promoter. The hygromycin resistance gene present inthe vector allows for selection of mammalian cells that express therecombinant protein, and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 151P3D4constitutively, 151P3D4 ORF, or portions thereof, of 151P3D4 are clonedinto pSRα constructs. Amphotropic and ecotropic retroviruses aregenerated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 151P3D4,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 151P3D4 sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 60) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs are made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6× Hisfusion proteins of the full-length 151P3D4 proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 151P3D4. High virus titerleading to high level expression of 151P3D4 is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.A 151P3D4 coding sequences or fragments thereof are amplified by PCR andsubcloned into the AdEasy shuttle vector (Stratagene). Recombination andvirus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 151P3D4coding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 151P3D4 inmammalian cells, coding sequences of 151P3D4, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant151P3D4. These vectors are thereafter used to control expression of151P3D4 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 151P3D4 proteins in a baculovirus expressionsystem, 151P3D4 ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-151P3D4 isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 151P3D4 protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant 151P3D4protein can be detected using anti-151P3D4 or anti-His-tag antibody.151P3D4 protein can be purified and used in various cell-based assays oras immunogen to generate polyclonal and monoclonal antibodies specificfor 151P3D4.

Example 9 Antigenicity Profiles and Secondary Structure

FIGS. 5(A & B), FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), andFIGS. 9(A & B) depict graphically five amino acid profiles of 151P3D4variants 1 and 2, each assessment available by accessing the ProtScalewebsite located on the World Wide Web at(.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biologyserver.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of the 151P3D4 protein. Each of the above amino acidprofiles of 151P3D4 were generated using the following ProtScaleparameters for analysis: 1) A window size of 9; 2) 100% weight of thewindow edges compared to the window center; and, 3) amino acid profilevalues normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profile, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the 151P3D4 variant proteins indicated, e.g., bythe profiles set forth in FIGS. 5(A & B), FIGS. 6(A & B), FIGS. 7(A &B), FIGS. 8(A & B), and/or FIGS. 9(A & B) are used to prepareimmunogens, either peptides or nucleic acids that encode them, togenerate therapeutic and diagnostic anti-151P3D4 antibodies. Theimmunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50contiguous amino acids, or the corresponding nucleic acids that encodethem, from the 151P3D4 protein variants 1 and 2 listed in FIGS. 2 and 3.In particular, peptide immunogens of the invention can comprise, apeptide region of at least 5 amino acids of FIGS. 2 and 3 in any wholenumber increment that includes an amino acid position having a valuegreater than 0.5 in the Hydrophilicity profiles of FIG. 5; a peptideregion of at least 5 amino acids of FIGS. 2 and 3 in any whole numberincrement that includes an amino acid position having a value less than0.5 in the Hydropathicity profile of FIG. 6; a peptide region of atleast 5 amino acids of FIGS. 2 and 3 in any whole number increment thatincludes an amino acid position having a value greater than 0.5 in thePercent Accessible Residues profiles of FIG. 7; a peptide region of atleast 5 amino acids of FIGS. 2 and 3 in any whole number increment thatincludes an amino acid position having a value greater than 0.5 in theAverage Flexibility profiles on FIG. 8; and, a peptide region of atleast 5 amino acids of FIGS. 2 and 3 in any whole number increment thatincludes an amino acid position having a value greater than 0.5 in theBeta-turn profile of FIG. 9. Peptide immunogens of the invention canalso comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 151P3D4 protein variants 1 and 2, namely thepredicted presence and location of alpha helices, extended strands, andrandom coils, is predicted from the primary amino acid sequence usingthe HNN—Hierarchical Neural Network method (Guermeur, 1997, Internetwebsite pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html),accessed from the ExPasy molecular biology server located on the WorldWide Web at (.expasy.ch/tools/). The analysis indicates that 151P3D4variant 1 is composed of 25.71% alpha helix, 21.47% extended strand, and52.82% random coil (FIG. 13A). Variant 2 is composed of 25.80% alphahelix, 16.64% extended strand, and 57.56% random coil (FIG. 13B).

Analysis for the potential presence of transmembrane domains in the151P3D4 variant proteins was carried out using a variety oftransmembrane prediction algorithms accessed from the ExPasy molecularbiology server located on the World Wide Web at (.expasy.ch/tools/). Theprograms do not predict the presence of transmembrane domains in the151P3D4 protein variants, suggesting that they are soluble proteins.

Example 10 Generation of 151P3D4 Polyclonal Antibodies Johnstone

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with a full length 151P3D4 protein variant,computer algorithms are employed in design of immunogens that, based onamino acid sequence analysis contain characteristics of being antigenicand available for recognition by the immune system of the immunized host(see the Example entitled “Antigenicity Profiles”). Such regions wouldbe predicted to be hydrophilic, flexible, in beta-turn conformations,and be exposed on the surface of the protein (see, e.g., FIGS. 5(A & B),FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), or FIGS. 9(A & B) foramino acid profiles that indicate such regions of 151P3D4 proteinvariants).

For example, recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of 151P3D4 proteinvariants are used as antigens to generate polyclonal antibodies in NewZealand White rabbits. For example, in 151P3D4 variant 1, such regionsinclude, but are not limited to, amino acids 99-151, amino acids218-249, and amino acids 311-332. In sequence specific for variant 2,such regions include, but are not limited to, amino acids 16-38, aminoacids 76-90, amino acids 182-230, and amino acids 383-400. It is usefulto conjugate the immunizing agent to a protein known to be immunogenicin the mammal being immunized. Examples of such immunogenic proteinsinclude, but are not limited to, keyhole limpet hemocyanin (KLH), serumalbumin, bovine thyroglobulin, and soybean trypsin inhibitor. In oneembodiment, a peptide encoding amino acids 311-332 of 151P3D4 variant 1is conjugated to KLH and used to immunize the rabbit. Alternatively theimmunizing agent may include all or portions of the 151P3D4 variantproteins, analogs or fusion proteins thereof. For example, the 151P3D4variant 1 amino acid sequence can be fused using recombinant DNAtechniques to any one of a variety of fusion protein partners that arewell known in the art, such as glutathione-S-transferase (GST) and HIStagged fusion proteins. Such fusion proteins are purified from inducedbacteria using the appropriate affinity matrix.

In one embodiment, a GST-fusion protein encoding the N-terminal regionof 151P3D4 variant 1, amino acids 16-150, minus the first 15 amino acidsthat likely encodes a cleavable signal peptide, is produced and purifiedand used as immunogen. Other recombinant bacterial fusion proteins thatmay be employed include maltose binding protein, LacZ, thioredoxin,NusA, or an immunoglobulin constant region (see the section entitled“Production of 151P3D4 in Prokaryotic Systems” and Current Protocols InMolecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds.,1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N.,and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).

In addition to bacterial derived fusion proteins, mammalian expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seethe section entitled “Production of Recombinant 151P3D4 in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, amino acids16-354 of variant 1, minus the endogenous signal peptide, is cloned intothe Tag5 mammalian secretion vector. The recombinant protein is purifiedby metal chelate chromatography from tissue culture supernatants of 293Tcells stably expressing the recombinant vector. The purified Tag5151P3D4 protein is then used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with the Tag5-151P3D4 variant 1 protein,the full-length 151P3D4 variant 1 cDNA is cloned into pCDNA 3.1 myc-hisexpression vector (Invitrogen, see the Example entitled “Production ofRecombinant 151P3D4 in Eukaryotic Systems”). After transfection of theconstructs into 293T cells, cell lysates are probed with theanti-151P3D4 serum and with anti-His antibody (Santa CruzBiotechnologies, Santa Cruz, Calif.) to determine specific reactivity todenatured 151P3D4 protein using the Western blot technique (FIG. 20)shows expression of Myc His epitope tagged 151P3D4 variant 1 protein in293T cells as detected by an anti-His antibody. In addition, the immuneserum is tested by fluorescence microscopy, flow cytometry andimmunoprecipitation against 293T and other recombinant151P3D4-expressing cells to determine specific recognition of nativeprotein. Western blot, immunoprecipitation, fluorescent microscopy, andflow cytometric techniques using cells that endogenously express 151P3D4are also carried out to test reactivity and specificity.

Anti-serum from rabbits immunized with 151P3D4 variant fusion proteins,such as GST and MBP fusion proteins, are purified by depletion ofantibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. For example, antiserum derivedfrom a GST-151P3D4 variant 1 fusion protein encoding amino acids 16-150is first purified by passage over a column of GST protein covalentlycoupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum isthen affinity purified by passage over a column composed of a MBP-fusionprotein also encoding amino acids 16-150 covalently coupled to Affigelmatrix. The serum is then further purified by protein G affinitychromatography to isolate the IgG fraction. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 11 Generation of 151P3D4 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 151P3D4 variants comprise thosethat react with epitopes specific for each variant protein or specificto sequences in common between the variants that would disrupt ormodulate the biological function of the 151P3D4 variants, for examplethose that would disrupt the interaction with ligands and bindingpartners. Immunogens for generation of such mAbs include those designedto encode or contain the entire 151P3D4 protein variant sequence,regions of the 151P3D4 protein variants predicted to be antigenic fromcomputer analysis of the amino acid sequence (see, e.g., FIGS. 5(A & B),FIGS. 6(A & B), FIGS. 7(A & B), FIGS. 8(A & B), or FIGS. 9(A & B), andthe Example entitled “Antigenicity Profiles”). Immunogens includepeptides, recombinant bacterial proteins, and mammalian expressed Tag 5proteins and human and murine IgG FC fusion proteins. In addition, cellsengineered to express high levels of a respective 151P3D4 variant, suchas 293T-151P3D4 variant 1 or 300.19-151P3D4 variant 1 murine Pre-Bcells, are used to immunize mice.

To generate mAbs to a 151P3D4 variant, mice are first immunizedintraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or10⁷ 151P3D4-expressing cells mixed in complete Freund's adjuvant. Miceare then subsequently immunized IP every 2-4 weeks with, typically,10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund'sadjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. Inaddition to the above protein and cell-based immunization strategies, aDNA-based immunization protocol is employed in which a mammalianexpression vector encoding a 151P3D4 variant sequence is used toimmunize mice by direct injection of the plasmid DNA. For example, aminoacids 16-354 is cloned into the Tag5 mammalian secretion vector and therecombinant vector is used as immunogen. In another example the sameamino acids are cloned into an Fc-fusion secretion vector in which the151P3D4 variant 1 sequence is fused at the amino-terminus to an IgKleader sequence and at the carboxyl-terminus to the coding sequence ofthe human or murine IgG Fc region. This recombinant vector is then usedas immunogen. The plasmid immunization protocols are used in combinationwith purified proteins expressed from the same vector and with cellsexpressing the respective 151P3D4 variant.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

In one embodiment for generating 151P3D4 monoclonal antibodies, aTag5-151P3D4 variant 1 antigen encoding amino acids 16-354, is expressedand purified from stably transfected 293T cells. Balb C mice areinitially immunized intraperitoneally with 25 μg of the Tag5-151P3D4variant 1 protein mixed in complete Freund's adjuvant. Mice aresubsequently immunized every two weeks with 25 μg of the antigen mixedin incomplete Freund's adjuvant for a total of three immunizations.ELISA using the Tag5 antigen determines the titer of serum fromimmunized mice. Reactivity and specificity of serum to full length151P3D4 variant 1 protein is monitored by Western blotting,immunoprecipitation and flow cytometry using 293T cells transfected withan expression vector encoding the 151P3D4 variant 1 cDNA (see e.g., theExample entitled “Production of Recombinant 151P3D4 in EukaryoticSystems” and FIG. 20. Other recombinant 151P3D4 variant 1-expressingcells or cells endogenously expressing 151P3D4 variant 1 are also used.Mice showing the strongest reactivity are rested and given a finalinjection of Tag5 antigen in PBS and then sacrificed four days later.The spleens of the sacrificed mice are harvested and fused to SPO/2myeloma cells using standard procedures (Harlow and Lane, 1988).Supernatants from HAT selected growth wells are screened by ELISA,Western blot, immunoprecipitation, fluorescent microscopy, and flowcytometry to identify 151P3D4 specific antibody-producing clones.

In another embodiment, a Tag5 antigen encoding amino acids 1-400 ofvariant 2 is produced, purified and used as immunogen to derivemonoclonal antibodies specific to 151P3D4 variant 2. Hybridomasupernatants are then screened on both 151P3D4 variant 2- and 151P3D4variant 1-expressing cells to identify specific anti-151P3D4 variant 2monoclonal antibodies.

The binding affinity of a 151P3D4 monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and are used to help define which 151P3D4monoclonal antibodies preferred for diagnostic or therapeutic use, asappreciated by one of skill in the art. The BIAcore system (Uppsala,Sweden) is a preferred method for determining binding affinity. TheBIAcore system uses surface plasmon resonance (SPR, Welford K. 1991,Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology295: 268) to monitor biomolecular interactions in real time. BIAcoreanalysis conveniently generates association rate constants, dissociationrate constants, equilibrium dissociation constants, and affinityconstants.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true K_(D) values.Peptide inhibitors are typically tested at concentrations ranging from120 μg/ml to 1.2 ng/ml, and are tested in two to four completelyindependent experiments. To allow comparison of the data obtained indifferent experiments, a relative binding figure is calculated for eachpeptide by dividing the IC₅₀ of a positive control for inhibition by theIC₅₀ for each tested peptide (typically unlabeled versions of theradiolabeled probe peptide). For database purposes, and inter-experimentcomparisons, relative binding values are compiled. These values cansubsequently be converted back into IC₅₀ nM values by dividing the IC₅₀nM of the positive controls for inhibition by the relative binding ofthe peptide of interest. This method of data compilation is accurate andconsistent for comparing peptides that have been tested on differentdays, or with different lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif- and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables V-XVIII andXXII-LI employ the protein sequence data from the gene product of151P3D4 set forth in FIGS. 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 151P3D4protein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or ΔG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:

“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . . ×a _(ni)

where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j_(i) to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of j_(i). For Class II peptides,if multiple alignments are possible, only the highest scoring alignmentis utilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 151P3D4 are scanned utilizing motifidentification software, to identify 8-, 9-10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 151P3D4 protein sequence(s) scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 Supermotif Bearing Epitopes

The 151P3D4 protein(s) scanned above is also analyzed for the presenceof 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC₅₀ of ≦500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the151P3D4 protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M-450)and the Detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8⁺ T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶ cells/ml. The magneticbeads are washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml Detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml β₂-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at ˜4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml β₂ microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 ng/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample−cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In Situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/ml. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4,200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25 μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×10⁴ CD8⁺ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 151P3D4. Briefly, PBMCs are isolated frompatients, re-stimulated with peptide-pulsed monocytes and assayed forthe ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2- and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analoged to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of5000 nM or less, to three of more A2 supertype alleles. The rationalefor this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with151P3D4-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 16 Identification and Confirmation of 151P3D4-Derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 151P3D4-derived, HLA class II HTL epitopes, a 151P3D4antigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, and three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 151P3D4-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 151P3D4-derived peptides found to bind common HLA-DR allelesare of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 151P3D4 antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 17 Immunogenicity of 151P3D4-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 151P3D4-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1−(1−Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%. An analogous approach can be used to estimatepopulation coverage achieved with combinations of class II motif-bearingepitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 19 CTL Recognition of Endogenously Processed Antigens afterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.1/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 151P3D4 expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 151P3D4 antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 20 Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 151P3D4-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 151P3D4-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/K^(b) mice, which are transgenic for the human HLA A2.1allele and are used to confirm the immunogenicity of HLA-A*0201 motif-or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously(base of the tail) with a 0.1 ml of peptide in Incomplete Freund'sAdjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello et al., J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be:[(1/50,000)−(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity.” Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a151P3D4-Specific Vaccine

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 151P3D4 clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 151P3D4. Forexample, if it has been observed that patients who spontaneously clear151P3D4-expressing cells generate an immune response to at least three(3) epitopes from 151P3D4 antigen, then at least three epitopes shouldbe included for HLA class I. A similar rationale is used to determineHLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site, at URL bimas.dcrt.nih.gov/.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 151P3D4, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected peptides, when administered,is safe, efficacious, and elicits an immune response similar inmagnitude to an immune response that controls or clears cells that bearor overexpress 151P3D4.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supermotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 151P3D4, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 151P3D4 to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the Ii protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the Ii protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to which it InducesImmunogenicity

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Barnett et al., Aids Res. and HumanRetroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci. USA 95:7648-53, 1998; Hanke andMcMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al.,Nature Med. 5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent151P3D4 expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 151P3D4-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against151P3D4-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native 151P3D4Sequences

A native 151P3D4 polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes can be used to generate a minigene construct. The construct isengineered to express the peptide, which corresponds to the nativeprotein sequence. The “relatively short” peptide is generally less than250 amino acids in length, often less than 100 amino acids in length,preferably less than 75 amino acids in length, and more preferably lessthan 50 amino acids in length. The protein sequence of the vaccinecomposition is selected because it has maximal number of epitopescontained within the sequence, i.e., it has a high concentration ofepitopes. As noted herein, epitope motifs may be nested or overlapping(i.e., frame shifted relative to one another). For example, withoverlapping epitopes, two 9-mer epitopes and one 10-mer epitope can bepresent in a 10 amino acid peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 151P3D4 antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally, such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup(s) that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 151P3D4, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 151P3D4 peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 151P3D4 and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 151P3D4 as well astumor-associated antigens that are often expressed with a target cancerassociated with 151P3D4 expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 151P3D4.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 151P3D4 HLA-A*0201-specific CTL frequencies from HLAA*0201-positive individuals at different stages of disease or followingimmunization comprising a 151P3D4 peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and β2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 151P3D4 epitope, andthus the status of exposure to 151P3D4, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from151P3D4-associated disease or who have been vaccinated with a 151P3D4vaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 151P3D4 vaccine.PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2 mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 μl/well ofcomplete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104,1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release−spontaneous release)/maximumrelease−spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 151P3D4 or a 151P3D4 vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 151P3D4 antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 29 Induction of Specific CTL Response in Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials in Patients Expressing 151P3D4

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses151P3D4. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTLs in cancer patients that express151P3D4, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTLs improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 151P3D4.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 151P3D4-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against151P3D4 is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 151P3D4 protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 151P3D4 antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 151P3D4. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et al., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 151P3D4 to isolatepeptides corresponding to 151P3D4 that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 151P3D4-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 151P3D4. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 151P3D4. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 151P3D4-encodingtranscript.

Example 35 Purification of Naturally-Occurring or Recombinant 151P3D4Using 151P3D4-Specific Antibodies

Naturally occurring or recombinant 151P3D4 is substantially purified byimmunoaffinity chromatography using antibodies specific for 151P3D4. Animmunoaffinity column is constructed by covalently coupling anti-151P3D4antibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin isblocked and washed according to the manufacturer's instructions.

Media containing 151P3D4 are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 151P3D4 (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/151P3D4 binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 36 Identification of Molecules which Interact with 151P3D4

151P3D4, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 151P3D4, washed, and anywells with labeled 151P3D4 complex are assayed. Data obtained usingdifferent concentrations of 151P3D4 are used to calculate values for thenumber, affinity, and association of 151P3D4 with the candidatemolecules.

Example 37 In Vivo Assay for 151P3D4 Tumor Growth Promotion

The effect of the 151P3D4 protein on tumor cell growth is evaluated invivo by evaluating tumor development and growth of cells expressing orlacking 151P3D4. For example, SCID mice are injected subcutaneously oneach flank with 1×10⁶ of either 3T3, bladder, kidney or ovary cancercell lines (e.g. SCABER, J82, PA-1, CaOv3, A498 or 769P cells)containing tkNeo empty vector or 151P3D4. At least two strategies may beused: (1) Constitutive 151P3D4 expression under regulation of a promotersuch as a constitutive promoter obtained from the genomes of virusessuch as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul.1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, aviansarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus andSimian Virus 40 (SV40), or from heterologous mammalian promoters, e.g.,the actin promoter or an immunoglobulin promoter, provided suchpromoters are compatible with the host cell systems, and (2) Regulatedexpression under control of an inducible vector system, such asecdysone, tetracycline, etc., provided such promoters are compatiblewith the host cell systems. Tumor volume is then monitored by calipermeasurement at the appearance of palpable tumors and followed over timeto determine if 151P3D4-expressing cells grow at a faster rate andwhether tumors produced by 151P3D4-expressing cells demonstratecharacteristics of altered aggressiveness (e.g. enhanced metastasis,vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1×10⁵ of the same cellsorthotopically to determine if 151P3D4 has an effect on local growth inthe bladder, kidney or ovary, and whether 151P3D4 affects the ability ofthe cells to metastasize, specifically to lymph nodes, adrenal, liverand bone (Miki T et al, Oncol Res. 2001; 12:209; Fu X et al, Int J.Cancer. 1991, 49:938; Kiguchi K et al, Clin Exp Metastasis. 1998,16:751).

The assay is also useful to determine the 151P3D4 inhibitory effect ofcandidate therapeutic compositions, such as for example, 151P3D4intrabodies, 151P3D4 antisense molecules and ribozymes.

Example 38 151P3D4 Monoclonal Antibody-Mediated Inhibition of Bladder,Kidney and Ovarian Tumors In Vivo

The significant expression of 151P3D4 in cancer tissues, together withits restrictive expression in normal tissues makes 151P3D4 a good targetfor antibody therapy. Similarly, 151P3D4 is a target for T cell-basedimmunotherapy. Thus, the therapeutic efficacy of anti-151P3D4 mAbs inhuman bladder cancer xenograft mouse models is evaluated by usingrecombinant cell lines such as SCABER-151P3D4, J82-151P3D4, and3T3-151P3D4 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1):p. 16-23). Similarly, anti-151P3D4 mAbs are evaluated in human kidneyand ovarian cancer xenograft models using recombinant cell lines such asA498-151P3D4 and PA1-151P3D4.

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in a mouse orthotopic bladder cancer xenograft model, a mousekidney cancer xenograft model and a mouse ovarian cancer xenograftmodel. The antibodies can be unconjugated, as discussed in this Example,or can be conjugated to a therapeutic modality, as appreciated in theart. Anti-151P3D4 mAbs inhibit formation of kidney, ovarian and bladderxenografts. Anti-151P3D4 mAbs also retard the growth of establishedorthotopic tumors and prolonged survival of tumor-bearing mice. Theseresults indicate the utility of anti-151P3D4 mAbs in the treatment oflocal and advanced stages of ovarian, kidney and bladder cancer. (See,e.g., Saffran, D., et al., PNAS 10:1073-1078 or the website located onthe World Wide Web at .pnas.org/cgi/doi/10.1073/pnas.051624698).

Administration of the anti-151P3D4 mAbs led to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 151P3D4 as anattractive target for immunotherapy and demonstrate the therapeuticpotential of anti-151P3D4 mAbs for the treatment of local and metastaticcancer. This example demonstrates that unconjugated 151P3D4 monoclonalantibodies are effective to inhibit the growth of human bladder, kidneyand ovarian tumor xenografts grown in SCID mice; accordingly acombination of such efficacious monoclonal antibodies is also effective.

Tumor Inhibition Using Multiple Unconjugated 151P3D4 mABs

Materials and Methods

151P3D4 Monoclonal Antibodies:

Monoclonal antibodies are raised against 151P3D4 as described in theExample entitled “Generation of 151P3D4 Monoclonal Antibodies (mAbs).”The antibodies are characterized by ELISA, Western blot, FACS, andimmunoprecipitation for their capacity to bind 151P3D4. Epitope mappingdata for the anti-151P3D4 mAbs, as determined by ELISA and Westernanalysis, recognize epitopes on the 151P3D4 protein. Immunohistochemicalanalysis of prostate cancer tissues and cells with these antibodies isperformed.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections ofSCABER, J82, A498, 769P, CaOv1 or PA1 tumor xenografts.

Cell Lines

The bladder, kidney and ovary carcinoma cell lines, SCABER, J82, A498,769P, CaOv1 and PA1 as well as the fibroblast line NIH 3T3 (AmericanType Culture Collection) are maintained in DMEM supplemented withL-glutamine and 10% FBS.

A SCABER-151P3D4, J82-151P3D4, A498-151P3D4, 769P-151P3D4,CaOv1-151P3D4, PA1-151P3D4 and 3T3-151P3D4 cell populations aregenerated by retroviral gene transfer as described in Hubert, R. S., etal., Proc Natl Acad Sci USA, 1999. 96(25): 14523.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ cancercells mixed at a 1:1 dilution with Matrigel (Collaborative Research) inthe right flank of male SCID mice. To test antibody efficacy on tumorformation, i.p. antibody injections are started on the same day astumor-cell injections. As a control, mice are injected with eitherpurified mouse IgG (ICN) or PBS; or a purified monoclonal antibody thatrecognizes an irrelevant antigen not expressed in human cells. Tumorsizes are determined by caliper measurements, and the tumor volume iscalculated as: Length×Width×Height. Mice with s.c. tumors greater than1.5 cm in diameter are sacrificed.

Orthotopic injections are performed under anesthesia by usingketamine/xylazine. For bladder orthotopic studies, an incision is madethrough the abdomen to expose the bladder, and tumor cells (5×10⁵) mixedwith Matrigel are injected into the bladder wall in a 10-μl volume. Tomonitor tumor growth, mice are palpated and blood is collected on aweekly basis to measure BTA levels. For kidney and ovary orthopoticmodels, an incision is made through the abdominal muscles to expose thekidney or the ovary. Tumor cells mixed with Matrigel are injected underthe kidney capsule or into the ovary in a 10-μl volume (Yoshida Y et al,Anticancer Res. 1998, 18:327; Ahn et al, Tumour Biol. 2001, 22:146). Tomonitor tumor growth, blood is collected on a weekly basis measuringG250 and SM047 levels. The mice are segregated into groups for theappropriate treatments, with anti-151P3D4 or control mAbs being injectedi.p.

Anti-151P3D4 mABs Inhibit Growth of 151P3D4-Expressing Xenograft-CancerTumors

The effect of anti-151P3D4 mAbs on tumor formation is tested on thegrowth and progression of bladder, kidney and ovarian cancer xenograftsusing UC3-151P3D4, J82-151P3D4, A498-151P3D4, 769P-151P3D4,CaOv1-151P3D4 and PA1-151P3D4 orthotopic models. As compared with thes.c. tumor model, the orthotopic model, which requires injection oftumor cells directly in the mouse bladder, kidney and ovary,respectively, results in a local tumor growth, development of metastasisin distal sites, deterioration of mouse health, and subsequent death(Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992.52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). Thefeatures make the orthotopic model more representative of human diseaseprogression and allowed us to follow the therapeutic effect of mAbs onclinically relevant end points.

Accordingly, tumor cells are injected into the mouse bladder, kidney orovary, and 2 days later, the mice are segregated into two groups andtreated with either: a) 200-500 μg, of anti-151P3D4 Ab, or b) PBS threetimes per week for two to five weeks.

A major advantage of the orthotopic cancer models is the ability tostudy the development of metastases. Formation of metastasis in micebearing established orthotopic tumors is studies by IHC analysis on lungsections using an antibody against a tumor-specific cell-surface proteinsuch as anti-CK20 for bladder cancer, anti-G250 for kidney cancer andSM047 antibody for ovarian cancer models (Lin S et al, Cancer DetectPrey. 2001; 25:202; McCluggage W et al, Histopathol 2001, 38:542).

Mice bearing established orthotopic tumors are administered 1000 μginjections of either anti-151P3D4 mAb or PBS over a 4-week period. Micein both groups are allowed to establish a high tumor burden, to ensure ahigh frequency of metastasis formation in mouse lungs. Mice then arekilled and their bladders, livers, bone and lungs are analyzed for thepresence of tumor cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-151P3D4antibodies on initiation and progression of prostate and kidney cancerin xenograft mouse models. Anti-151P3D4 antibodies inhibit tumorformation of tumors as well as retarding the growth of alreadyestablished tumors and prolong the survival of treated mice. Moreover,anti-151P3D4 mAbs demonstrate a dramatic inhibitory effect on the spreadof local bladder, kidney and ovarian tumor to distal sites, even in thepresence of a large tumor burden. Thus, anti-151P3D4 mAbs areefficacious on major clinically relevant end points (tumor growth),prolongation of survival, and health.

Example 39 Therapeutic and Diagnostic Use of Anti-151P3D4 Antibodies inHumans

Anti-151P3D4 monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-151P3D4 mAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of 151P3D4 in carcinoma and in metastaticdisease demonstrates the usefulness of the mAb as a diagnostic and/orprognostic indicator. Anti-151P3D4 antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-151P3D4 mAb specifically binds tocarcinoma cells. Thus, anti-151P3D4 antibodies are used in diagnosticwhole body imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastaticcancers that exhibit expression of 151P3D4. Shedding or release of anextracellular domain of 151P3D4 into the extracellular milieu, such asthat seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology27:563-568 (1998)), allows diagnostic detection of 151P3D4 byanti-151P3D4 antibodies in serum and/or urine samples from suspectpatients.

Anti-151P3D4 antibodies that specifically bind 151P3D4 are used intherapeutic applications for the treatment of cancers that express151P3D4. Anti-151P3D4 antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-151P3D4 antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “151P3D4 Monoclonal Antibody-mediatedInhibition of Bladder and Lung Tumors In Vivo”). Conjugated andunconjugated anti-151P3D4 antibodies are used as a therapeutic modalityin human clinical trials either alone or in combination with othertreatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through Use of Human Anti-151P3D4 Antibodies In Vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on 151P3D4, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 151P3D4 expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-151P3D4 antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-151P3D4 antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-151P3D4 antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-151P3D4antibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine oryttrium (I¹³¹, Y⁹⁰) to anti-151P3D4 antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 151P3D4. In connection with theuse of the anti-151P3D4 antibodies as imaging agents, the antibodies areused as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In) 151P3D4antibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses 151P3D4 (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-151P3D4 antibodies can beadministered with doses in the range of 5 to 400 mg/m², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-151P3D4 antibodies relative to the affinity of a known antibody forits target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-151P3D4 antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-151P3D4 antibodies can be lower, perhaps in the range of 50to 300 mg/m², and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-151P3D4 antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-151P3D4antibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-151P3D4antibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 151P3D4 expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 151P3D4.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-151P3D4 antibodies are found to be safe upon humanadministration.

Example 41 Human Clinical Trial Adjunctive Therapy with HumanAnti-151P3D4 Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-151P3D4 antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-151P3D4antibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent, such as cisplatin, topotecan, doxorubicin,adriamycin, taxol, or the like, is assessed. The trial design includesdelivery of six single doses of an anti-151P3D4 antibody with dosage ofantibody escalating from approximately about 25 mg/m² to about 275 mg/m²over the course of the treatment in accordance with the followingschedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + + (standarddose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i) cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 151P3D4.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-151P3D4 antibodies are demonstrated to be safe and efficacious,Phase II trials confirm the efficacy and refine optimum dosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-151P3D4Antibody

Anti-151P3D4 antibodies are safe in connection with the above-discussedadjunctive trial, a Phase II human clinical trial confirms the efficacyand optimum dosing for monotherapy. Such trial is accomplished, andentails the same safety and outcome analyses, to the above-describedadjunctive trial with the exception being that patients do not receivechemotherapy concurrently with the receipt of doses of anti-151P3D4antibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-151P3D4Antibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-151P3D4 antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 151P3D4 to Known Sequences

Two variants of 151P3D4 have been identified, 151P3D4 v.1 and v 2. The151P3D4 v.1 gene exhibits strong homology to a previously cloned gene,namely the human cartilage linking protein 1 (gi 4503053), and shows100% identity to that gene over the entire length of the protein (FIG.4B). In addition, the 151P3D4 v.1 protein shows homology to the bovineand rat homologs of the human cartilage linking protein (gi 1709660 andgi 9506519) (FIGS. 4F and 4G). 151P3D4 v.1 is a 354 aa protein whichlocalizes primarily to the extracellular compartment (see Table XXI).The second variant, 151P3D4 v.2, is a 721 aa protein, that sharesidentity with 151P3D4 v.1 over 200 amino acids (Table LV and FIG. 4D).The 151P3D4 v.2 gene also exhibits homology to the human cartilage linkprotein-1 (gi 4503053), showing 99% identity and 99% homology to thatprotein (FIG. 4H). However, this homology between variant 2 andcartilage link protein does not extend over the entire length of variant2, but is limited to the last 400 aa of that protein. The first 400 aaof 151P3D4 v.2 show homology to human ribosomal protein L13a of the 60Ssubunit (gi. 18574549) (see Table XXI). Besides the addition of 400 aaat its N-terminus, 151P3D4 v.2 also differs from variant 1 in itslocalization profile. 151P3D4 v.2 localizes to the cytosol, withpotential localization to the nucleus (see Table XXI). Motif analysisrevealed the presence of link motif as well as immunoglobulin domain inboth 151P3D4 variants (see Table XXI).

Cartilage link protein-1, a protein with a known link motif, has beenshown to regulate tissue remodeling, bone resorption and proteininteraction (Chen Q et al. Dev Biol. 1995, 172:293). The importance ofcartilage link protein 1 is illustrated in engineered mice lackingcartilage link protein (Watanabe H, Yamada Y. Nat. Genet. 1999, 21:225).These mutant mice demonstrate defects in cartilage and bone development.The cartilage link protein, via its link motif, mediates cell adhesionof fibroblasts and other cells to extracellular matrix (Yang B et al,Matrix Biol. 1998, 16:541). The link motif is a binding domain forhyaluronic acid (Kohda D et al, Cell. 1996, 86:767), with a structurevery similar to type C-lectin. It plays a role in the assembly ofextracellular matrix, cell adhesion, and migration (Kohda D et al, Cell.1996, 86:767). The immunoglobulin domain is a 100 aa long motif whichincludes a conserved intra-domain disulfide bond. Immunoglobulin-likedomains participate in protein interactions (Wang J, Springer T A.Immunol Rev. 1998, 163:197).

The presence of an immunoglobulin motif and a link motif indicate that151P3D4 regulates protein interactions and participates in the processof cell adhesion, cell migration, tumor formation and progression. Byway of its protein interaction domain, 151P3D4 functions in regulatingsignal transduction in mammalian cells, thereby regulating geneexpression and cellular outcomes, including cell proliferation,survival, invasion, motility, etc, all of which have a direct effect ontumor growth and progression.

Accordingly, when 151P3D4 functions as a regulator of proteininteractions, cell adhesion, tumor formation, invasion or cellsignaling, 151P3D4 is used for therapeutic, diagnostic, prognosticand/or preventative purposes. In addition, when a variant of 151P3D4 isexpressed in cancerous tissues, such as those listed in Table I, theyare used for therapeutic, diagnostic, prognostic and/or preventativepurposes.

Example 45 Regulation of Transcription

The localization of 151P3D4 coupled to the presence of proteininteraction domains within its sequence, indicate that 151P3D4 modulatesthe transcriptional regulation of eukaryotic genes. Regulation of geneexpression is confirmed, e.g., by studying gene expression in cellsexpressing or lacking 151P3D4. For this purpose, two types ofexperiments are performed.

In the first set of experiments, RNA from parental and151P3D4-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer.2000. 83:246). Resting cells as well as cells treated with FBS, androgenor growth factors are compared. Differentially expressed genes areidentified in accordance with procedures known in the art. Thedifferentially expressed genes are then mapped to biological pathways(Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation.

Thus, 151P3D4 plays a role in gene regulation, and it is used as atarget for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Example 46 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). In particular, protein interaction motifshave been instrumental in inducing kinase activation, recruitment ofproteins and complex formation (Samelson L. Annu Rev Immunol. 2002;20:371). Based on the presence of a protein interaction motif, 151P3D4regulates signaling pathways important for cell growth and invasion. Inaddition, the 151P3D4 protein contains several phosphorylation sites(see Table XX) indicating an association with specific signalingcascades. Using immunoprecipitation and Western blotting techniques,proteins are identified that associate with 151P3D4 and mediatesignaling events. Several pathways known to play a role in cancerbiology can be regulated by 151P3D4, including phospholipid pathwayssuch as PI3K, AKT, etc, adhesion and migration pathways, including FAK,Rho, Rac-1, β-catenin, etc, as well as mitogenic/survival cascades suchas ERK, p38, etc (Cell Growth Differ. 2000, 11:279; J Biol. Chem. 1999,274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.).

To confirm that 151P3D4 directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress

2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation

3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress

4. ARE-luc, androgen receptor; steroids/MAPK;growth/differentiation/apoptosis

5. p53-luc, p53; SAPK; growth/differentiation/apoptosis

6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

7. TCF-luc, TCF/Lef; β-catenin, Adhesion/invasion

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by 151P3D4 are mapped and used for theidentification and validation of therapeutic targets. When 151P3D4 isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Tumor Progression

Based on the role of link motif in cell adhesion, cell migration andtumor formation, the 151P3D4 gene can contribute to tumor initiation andprogression. The role of 151P3D4 in tumor growth is confirmed in avariety of primary and transfected cell lines including bladder, kidneyand ovary cell lines, as well as NIH 3T3 cells engineered to stablyexpress 151P3D4. Parental cells lacking 151P3D4 and cells expressing151P3D4 are evaluated for cell growth using a well-documentedproliferation assay (Fraser S P, Grimes J A, Djamgoz M B. Prostate.2000; 44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996,7:288).

To confirm the role of 151P3D4 in the transformation process, its effectin colony forming assays is investigated. Parental NIH-3T3 cells lacking151P3D4 are compared to NIH-3T3 cells expressing 151P3D4, using a softagar assay under stringent and more permissive conditions (Song Z. etal. Cancer Res. 2000; 60:6730).

To confirm the role of 151P3D4 in invasion and metastasis of cancercells, a well-established assay is used, e.g., a Transwell Insert Systemassay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells,including bladder, ovary and kidney cell lines lacking 151P3D4 arecompared to cells expressing 151P3D4. Cells are loaded with thefluorescent dye, calcein, and plated in the top well of the Transwellinsert coated with a basement membrane analog. Invasion is determined byfluorescence of cells in the lower chamber relative to the fluorescenceof the entire cell population.

151P3D4 can also play a role in cell cycle and apoptosis. Parental cellsand cells expressing 151P3D4 are compared for differences in cell cycleregulation using a well-established BrdU assay (Abdel-Malek Z A. J CellPhysiol. 1988, 136:247). In short, cells are grown under both optimal(full serum) and limiting (low serum) conditions are labeled with BrdUand stained with anti-BrdU Ab and propidium iodide. Cells are analyzedfor entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in controlparental cells and cells expressing 151P3D4, including normal and tumorbladder, kidney and ovary cells. Engineered and parental cells aretreated with various chemotherapeutic agents, such as etoposide, taxol,etc, and protein synthesis inhibitors, such as cycloheximide. Cells arestained with annexin V-FITC and cell death is measured by FACS analysis.The modulation of cell death by 151P3D4 can play a critical role inregulating tumor progression and tumor load.

When 151P3D4 plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 48 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J.Endocrinology. 1998 139:441). Based on the effect of phosphodiesteraseinhibitors on endothelial cells, 151P3D4 plays a role in angiogenesis(DeFouw L et al, Microvasc Res 2001, 62:263). Several assays have beendeveloped to measure angiogenesis in vitro and in vivo, such as thetissue culture assays endothelial cell tube formation and endothelialcell proliferation. Using these assays as well as in vitroneo-vascularization, the role of 151P3D4 in angiogenesis, enhancement orinhibition, is confirmed.

For example, endothelial cells engineered to express 151P3D4 areevaluated using tube formation and proliferation assays. The effect of151P3D4 is also confirmed in animal models in vivo. For example, cellseither expressing or lacking 151P3D4 are implanted subcutaneously inimmunocompromised mice. Endothelial cell migration and angiogenesis areevaluated 5-15 days later using immunohistochemistry techniques. 151P3D4affects angiogenesis, and it is used as a target for diagnostic,prognostic, preventative and/or therapeutic purposes

Example 49 Involvement in Protein-Protein Interactions

Link as well as immunoglobulin motifs have been shown to mediateinteraction with other proteins, resulting in the formation of amulti-protein complex ( ). Using immunoprecipitation techniques as wellas two yeast hybrid systems, proteins are identified that associate with151P3D4. Immunoprecipitates from cells expressing 151P3D4 and cellslacking 151P3D4 are compared for specific protein-protein associations.

Studies are performed to confirm the extent of association of 151P3D4with effector molecules, such as nuclear proteins, transcriptionfactors, kinases, phosphates etc. Studies comparing 151P3D4 positive and151P3D4 negative cells as well as studies comparing unstimulated/restingcells and cells treated with epithelial cell activators, such ascytokines, growth factors and anti-integrin Ab reveal uniqueinteractions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodology (Curr Opin Chem. Biol. 1999, 3:64). A vector carryinga library of proteins fused to the activation domain of a transcriptionfactor is introduced into yeast expressing a 151P3D4-DNA-binding domainfusion protein and a reporter construct. Protein-protein interaction isdetected by colorimetric reporter activity. Specific association witheffector molecules and transcription factors directs one of skill to themode of action of 151P3D4, and thus identifies therapeutic, prognostic,preventative and/or diagnostic targets for cancer. This and similarassays are also used to identify and screen for small molecules thatinteract with 151P3D4.

Thus it is found that 151P3D4 associates with proteins and smallmolecules. Accordingly, 151P3D4 and these proteins and small moleculesare used for diagnostic, prognostic, preventative and/or therapeuticpurposes.

Example 50 Involvement in Adhesion

Cell adhesion plays a critical role in tissue colonization andmetastasis. The presence of link motif in 151P3D4 is indicative of itsrole in cell adhesion. To confirm that 151P3D4 plays a role in celladhesion, control cells lacking 151P3D4 are compared to cells expressing151P3D4, using techniques previously described (see, e.g., Haier et al,Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst.1998, 90:118). Briefly, in one embodiment, cells labeled with afluorescent indicator, such as calcein, are incubated on tissue culturewells coated with media alone or with matrix proteins. Adherent cellsare detected by fluorimetric analysis and percent adhesion iscalculated. This experimental system can be used to identify proteins,antibodies and/or small molecules that modulate cell adhesion toextracellular matrix and cell-cell interaction. Since cell adhesionplays a critical role in tumor growth, progression, and, colonization,the gene involved in this process can serves as a diagnostic,preventative and therapeutic modality.

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

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LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100168214A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An isolated polynucleotide, comprising the nucleotide sequence of SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 or SEQ ID NO: 22.2. The isolated polynucleotide of claim 1, the nucleotide sequence ofwhich is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10,12, 14, 16, 18, 20, and
 22. 3. A pharmaceutical composition, comprisingthe isolated polynucleotide of claim 1 and a pharmaceutically acceptablecarrier.
 4. A recombinant viral expression vector, comprising theisolated polynucleotide of claim
 1. 5. The recombinant viral expressionvector of claim 4, wherein the viral expression vector is selected fromthe group consisting of vaccinia, fowlpox, canarypox, adenovirus,influenza, poliovirus, adeno-associated virus, lentivirus, and Sindbisvirus vectors.
 6. An isolated host cell that contains the expressionvector of claim
 5. 7. A process for producing a protein, comprising:culturing the isolated host cell of claim 6 under conditions sufficientfor the production of a protein from the expression vector, andrecovering the protein from the culture.
 8. The process of claim 8,wherein the protein is recovered using chromatography.
 9. An isolatedpolynucleotide, comprising: (a) the sequence of SEQ ID NO:4, fromnucleotide residue numbers 1 through 2166; (b) the sequence of SEQ IDNO:12, from nucleotide residue numbers 316 through 1380; or (c) thesequence of SEQ ID NO:14, from nucleotide residue numbers 316 through1380.
 10. A pharmaceutical composition, comprising the isolatedpolynucleotide of claim 9 and a pharmaceutically acceptable carrier. 11.A recombinant expression vector, comprising the isolated polynucleotideof claim 9, wherein the vector is a viral vector.
 12. The recombinantexpression vector of claim 11, wherein the viral vector is selected fromthe group consisting of vaccinia, fowlpox, canarypox, adenovirus,influenza, poliovirus, adeno-associated virus, lentivirus, and Sindbisvirus.
 13. An isolated host cell that contains the expression vector ofclaim
 12. 14. A process for producing a protein, comprising: culturingthe isolated host cell of claim 13 under conditions sufficient for theproduction of a protein from the expression vector, and recovering theprotein from the culture.
 15. The process of claim 14, wherein theprotein is recovered using chromatography.